Methods and compositions for highly sensitive detection of molecules

ABSTRACT

Disclosed are methods, kits, and compositions for the highly sensitive detection of molecules. The methods, kits, and compositions are useful in determining concentrations of molecules in samples to levels of 1 femtomolar, 1 attomolar, or lower. The methods, kits, and compositions also allow the determination of concentration over a wide range, e.g., 7-log range, without need for sample dilution.

CROSS-REFERENCE

This application is a continuation-in-part application of Ser. No.11/048,660, filed Jan. 28, 2005, which is incorporated herein byreference in its entirety and to which application we claim priorityunder 35 USC §120. Application Ser. No. 11/048,660 claims the benefit ofU.S. Provisional Application No. 60/613,881, filed Sep. 28, 2004, U.S.Provisional Application No. 60/624,785, filed Oct. 27, 2004, and U.S.Provisional Application No. 60/636,158, filed Dec. 13, 2004.

This application also claims priority under 35 USC §119 to U.S.Provisional Application No. 60/789,304, filed Apr. 4, 2006, U.S.Provisional Application No. 60/793,664, filed Apr. 19, 2006, U.S.Provisional Application No. 60/808,662, filed May 26, 2006, U.S.Provisional Application No. 60/861,498, filed Nov. 28, 2006, and U.S.Provisional Application No. 60/872,986, filed Dec. 4, 2006, all of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Advances in biomedical research, medical diagnosis, prognosis,monitoring and treatment selection, bioterrorism detection, and otherfields involving the analysis of multiple samples of low volume andconcentration of analytes have led to development of sample analysissystems capable of sensitively detecting particles in a sample atever-decreasing concentrations. U.S. Pat. Nos. 4,793,705 and 5,209,834describe previous systems in which extremely sensitive detection hasbeen achieved. The present invention provides further development inthis field.

SUMMARY OF THE INVENTION

In one aspect the invention involves an apparatus.

In some embodiments of the apparatus, the invention involves an analyzersystem kit for detecting a single protein molecule in a sample, the kitcomprising an analyzer and at least one label comprising a fluorescentmoiety and a binding partner for the protein molecule, and where theanalyzer contains: a) an electromagnetic radiation source forstimulating the fluorescent moiety; b) a capillary flow cell for passingthe label; c) a source of motive force for moving the label in thecapillary flow cell; d) an interrogation space defined within thecapillary flow cell for receiving electromagnetic radiation emitted fromthe electromagnetic source; and e) an electromagnetic radiation detectoroperably connected to the interrogation space for measuring anelectromagnetic characteristic of the stimulated fluorescent moiety;where the fluorescent moiety is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules. In some embodiments of the apparatus of the invention, theanalyzer system comprises not more than one interrogation space. In someembodiments of the apparatus of the invention, the electromagneticradiation source is a laser, and where the laser has a power output ofat least about 3, 5, 10, or 20 mW. In some embodiments of the apparatusof the invention, the fluorescent moiety involves a fluorescentmolecule. In some embodiments, the fluorescent molecule is a dyemolecule. In some embodiments, the dye molecule include at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. In some embodiments, the fluorescent moiety is aquantum dot. In some embodiments of the apparatus of the invention, theelectromagnetic radiation source is a continuous wave electromagneticradiation source. In some embodiments, the continuous waveelectromagnetic radiation source is a light-emitting diode or acontinuous wave laser. In some embodiments of the apparatus of theinvention, the motive force is pressure. In some embodiments of theapparatus of the invention, the detector is an avalanche photodiodedetector.

In some embodiments of the apparatus of the invention, the analyzersystem further include a confocal optical arrangement for deflecting alaser beam onto the interrogation space and for imaging the stimulateddye molecule, where the confocal optical arrangement comprises anobjective lens having a numerical aperture of at least about 0.8. Insome embodiments of the apparatus of the invention, the analyzer systemfurther includes a sampling system capable of automatically sampling aplurality of samples and providing a fluid communication between asample container and the interrogation space. In some embodiments of theapparatus of the invention, the analyzer system further includes asample recovery system in fluid communication with the interrogationspace, where the recovery system is capable of recovering substantiallyall of the sample.

In some embodiments the invention includes an analyzer for determiningthe concentration of species in a sample, where the analyzer is capableof determining the concentration over a dynamic range of concentrationsof at least about 10⁵. In some embodiments, the dynamic range is fromabout 1 femtomolar to about 100 picomolar.

In another aspect the invention includes methods.

In some embodiments the invention include a method for determining thepresence or absence of a single molecule of a protein in a biologicalsample, including labeling the molecule with a label and detecting thepresence or absence of the label in a single molecule detector, wherethe label includes a fluorescent moiety that is capable of emitting atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and where the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments the single moleculedetector comprises not more than one interrogation space. In someembodiments of the methods of the invention, the limit of detection ofthe single molecule in the sample is less than about 10, 1, 0.1, 0.01,or 0.001 femtomolar. In some embodiments, the limit of detection is lessthan about 1 femtomolar. In some embodiments of the methods of theinvention, detecting the presence or absence of the label in a singlemolecule detector involves detecting electromagnetic radiation emittedby the fluorescent moiety. In some embodiments of the methods of theinvention, the methods further include exposing the fluorescent moietyto electromagnetic radiation. In some embodiments, the electromagneticradiation is provided by a laser. In some embodiments, the laserstimulates the moiety at a power output of less than about 20 mW. Insome embodiments, the laser stimulates the moiety for a duration of lessthan about 1000, 250, 100, 50, 25 or 10 microseconds. In someembodiments of the methods of the invention, the label further includesa binding partner specific for binding the molecule. In someembodiments, the binding partner is an antibody. In some embodiments ofthe methods of the invention, the fluorescent moiety comprises afluorescent dye molecule. In some embodiments, the dye molecule includesat least one substituted indolium ring system in which the substituenton the 3-carbon of the indolium ring contains a chemically reactivegroup or a conjugated substance. In some embodiments, the dye moleculeis an AlexFluor molecule selected from the group consisting ofAlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 orAlexaFluor 700. In some embodiments, the dye molecule is anAlexaFluor647 dye molecule. In some embodiments, the fluorescent moietycomprises a plurality of AlexaFluor 647 molecules. In some embodiments,the plurality of AlexaFluor 647 molecules comprises about 2-4 AlexaFluor647 molecules. In some embodiments, the fluorescent moiety is a quantumdot. In some embodiments of the methods of the invention, the methodsfurther include measuring the concentration of the protein in thesample. In some embodiments of the methods of the invention, thedetecting the presence or absence of the label includes: (i) passing aportion of the sample through an interrogation space; and (ii)subjecting the interrogation space to exposure to electromagneticradiation, the electromagnetic radiation being sufficient to stimulatethe fluorescent moiety to emit photons, if the label is present; and(iii) detecting photons emitted during the exposure of step (ii). Insome embodiments of the methods of the invention, the methods furtherincludes determining a background photon level in the interrogationspace, where the background level represents the average photon emissionof the interrogation space when it is subjected to electromagneticradiation in the same manner as in step (ii), but without label in theinterrogation space. In some embodiments of the methods of theinvention, the method further includes comparing the amount of photonsdetected in step (iii) to a threshold photon level, where the thresholdphoton level is a function of the background photon level, where anamount of photons detected in step (iii) greater that the thresholdlevel indicates the presence of the label, and an amount of photonsdetected in step (iii) equal to or less than the threshold levelindicates the absence of the label.

In some embodiments the invention involve a method for determining thepresence or absence of a biological state in a subject, including: a)performing an immunoassay on a sample from the subject, where theimmunoassay comprises binding a plurality of labels for a marker to aplurality of molecules of the marker in the sample, where the label isspecific to the marker and where one label binds to one molecule ofmarker, and where the label comprises an antibody attached to afluorescent moiety that is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules; b) detecting the labels where the detecting comprisesdetecting single labels with a single molecule detector; and c)determining a concentration for the marker in the sample based on thenumber of labels detected in step b). In some embodiments of the methodsof the invention, the method further include comparing the concentrationobtained in step b) to a concentration or range of concentrations forthe marker that are known to be indicative of the presence or absence ofthe biological state. In some embodiments of the methods of theinvention, the subject is a human. In some embodiments, the sample isblood, serum, plasma, urine or exhaled breath condensate.

In another aspect the invention provides compositions.

In some embodiments the invention provides a label for detecting abiological molecule comprising a binding partner for the biologicalmolecule that is attached to a fluorescent moiety, where the fluorescentmoiety is capable of emitting at least about 200 photons when simulatedby a laser emitting light at the excitation wavelength of the moiety,where the laser is focused on a spot not less than about 5 microns indiameter that contains the moiety, and where the total energy directedat the spot by the laser is no more than about 3 microJoules, where themoiety comprises about 2 to 4 fluorescent entities. In some embodimentsof the composition of the invention, the biological molecule is aprotein or a small molecule. In some embodiments, the biologicalmolecule is a protein. In some embodiments, the fluorescent entitiescomprise fluorescent dye molecules. In some embodiments, the fluorescentdye molecules include at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the dye molecules are AlexFluor molecules that can beAlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 orAlexaFluor 700. In some embodiments, the dye molecules are AlexaFluor647dye molecules. In some embodiments of the methods of the invention, thedye molecules comprise a first type and a second type of dye molecules.In some embodiments, the first type and second type of dye moleculeshave different emission spectra. In some embodiments, the ratio of thenumber of first type to second type of dye molecule is 4:1, 3:1, 2:1,1:1, 1:2, 1:3 or 1:4. In some embodiments of the methods of theinvention, the binding partner is an antibody.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B. Schematic diagram of the arrangement of the componentsof a single particle analyzer. FIG. 3A shows an analyzer that includesone electromagnetic source and one electromagnetic detector; FIG. 3Bshows an analyzer that includes two electromagnetic sources and oneelectromagnetic detectors.

FIGS. 2A and 2B. Schematic diagrams of a capillary flow cell for asingle particle analyzer. FIG. 4A shows the flow cell of an analyzerthat includes one electromagnetic source; and FIG. 4B shows the flowcell of an analyzer that includes two electromagnetic sources.

FIGS. 3A and 3B. Schematic diagrams showing the conventional (A) andconfocal (B) positioning of laser and detector optics of a singleparticle analyzer. FIG. 5A shows the arrangement for an analyzer thathas one electromagnetic source and one electromagnetic detector; FIG. 5Bshows the arrangement for an analyzer that has two electromagneticsources and two electromagnetic detectors.

FIG. 4. Linearized standard curve for the range concentrations of cTnI.

FIG. 5. Biological threshold (cutoff concentration) for cTnI is at acTnI concentration of 7 pg/ml, as established at the 99th percentilewith a corresponding CV of 10%.

FIG. 6. Correlation of assay results of cTnI determined using theanalyzer system of the invention with standard measurements provided bythe National Institute of Standards and Technology (R2=0.9999).

FIG. 7. Detection of cTnI in serial serum samples from patients whopresented at the emergency room with chest pain. The measurements madewith the analyzer system of the invention were compared to measurementsmade with a commercially available assay.

FIG. 8. Distribution of normal biological concentrations of cTnI andconcentrations of cTnI in serum samples from patients presenting withchest pain

FIG. 9. Competition curve for LTE4. The LOD was determined to be 1.5pg/ml LTE4.

FIG. 10. Graph showing the standard curve for concentrations of Akt1.The LOD was calculated to be 25 pg/ml Akt1.

FIG. 11. Graph showing the standard curve for concentrations of TGFβ.The LOD was calculated to be 350 pg/ml Akt1.

FIG. 12 A schematic representation of a kit that includes an analyzersystem for detecting a single protein molecule in a sample and least onelabel that includes a fluorescent moiety and a binding partner for theprotein molecule, where the analyzer includes an electromagneticradiation source for stimulating the fluorescent moiety; a capillaryflow cell for passing the label; a source of motive force for moving thelabel in the capillary flow cell; an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source; and an electromagnetic radiationdetector operably connected to the interrogation space for measuring anelectromagnetic characteristic of the stimulated fluorescent moiety,where the fluorescent moiety is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules

FIG. 13: Standard curve of TREM-1 measured in a sandwich moleculeimmunoassay developed for the single particle analyzer system. Thelinear range of the assay is 100-1500 fM.

FIG. 14 A-F. Detection of IL-6 and IL-8. A-B) Standard curve for IL-6.A) IL-6 standards, diluted according to a commercially available kit (R& D Systems, Minneapolis, Minn.) gave a linear response between 0.1 and10 pg/ml. B) IL-6 standard curve below 1 pg/ml. C) Distribution of IL-6and IL-8 identified in blood bank donor EDTA specimens. D) Range ofdetection at low concentrations of any analyte can be extended to higherconcentrations by switching the detection of the analyzer from countingmolecules (digital signal) to detecting the sum of photons (analogsignal) that are generated at the higher concentrations of analyte. Thesingle particle analyzer has an expanded linear dynamic range of 6 logs.E) Six-log range of detection based on switching from digital to analogdetection. F) Non-linearized standard curve showing range of lowconcentrations of IL-6 (0.1 fg/ml-10 fg/ml) determined by countingphotons emitted by individual particles (digital signal), and higherrange of concentrations of IL-6 (10 fg/ml-1 pg/ml).

FIG. 15. Comparison of assays of the invention with conventional assays.

DETAILED DESCRIPTION OF THE INVENTION

Outline

I. Introduction

II. Molecules for Sensitive Detection By the Methods and Compositions ofthe Invention

A. General

B. Markers

III. Labels

A. Binding partners

-   -   1. Antibodies

B. Fluorescent Moieties

-   -   1. Dyes    -   2 Quantum dots

C. Binding Partner-Fluorescent Moiety Compositions

IV. Highly Sensitive Analysis of Molecules

A. Sample

B. Sample preparation

C. Detection of molecule of interest and determination of concentration

V. Instruments and Systems Suitable for Highly Sensitive Analysis ofMolecules

A. Apparatus/System

B. Single Particle Analyzer

-   -   1 Electromagnetic Radiation Source    -   2. Capillary Flow Cell    -   3. Motive Force    -   4. Detectors

C. Sampling System

D. Sample preparation system

E. Sample recovery

VI. Methods Using Highly Sensitive Analysis of Molecules

A. Methods

B. Exemplary Markers

-   -   1. Cardiac damage    -   2. Infectious Diseases    -   3. Cytokines    -   4. Inflammatory Markers    -   5. Markers for a Disease State (Arthritis)    -   6. TGFβ    -   7. Akt1    -   8. Fas ligand

C. Business Methods

VII. Kits

I. Introduction

The invention provides instruments, kits, compositions, and methods forthe highly sensitive detection of single molecules, and for thedetermination of the concentration of the molecules in a sample. Thesensitivity and precision of the instruments, compositions, methods, andkits of the invention may be achieved in some embodiments by acombination of factors selected from, but not limited to,electromagnetic sources of appropriate wavelength and power output,appropriate interrogation space size, high numerical aperture lenses,detectors capable of detecting single photons, and data analysis systemsfor counting single molecules. The instruments of the invention arereferred to as “single molecule detectors” or “single particledetectors,” and are also encompassed by the terms “single moleculeanalyzers” and “single particle analyzers.” The sensitivity andprecision of the kits and methods of the invention are achieved in someembodiments by the use of the instruments of the invention together withby a combination of factors selected from, but not limited to, labelsfor molecules that exhibit characteristics that allow the molecules tobe detected at the level of the single molecule, and methods assayingthe label in the instruments described herein.”

The instruments, kits, and methods of the invention are especiallyuseful in the sensitive and precise detection of single proteinmolecules or small molecules, and for the determination of theconcentration of said molecules in a sample.

Thus the invention provides, in some embodiments, instruments and kitsfor the sensitive detection and determination of concentration ofmolecules by detection of single molecules, labels for such detectionand determination, and methods using such instruments and labels in theanalysis of samples. In particular, the sensitivity and precision of theinstruments, kits, and methods of the invention make possible thedetection and determination of concentration of molecules, e.g., markersfor biological states, at extremely low concentrations, e.g.,concentrations below about 100, 10, 1, 0.1, 0.01, or 0.001 femtomolar.In further embodiments, the instruments and kits of the invention arecapable of determining a concentration of a species, e.g., molecule, ina sample over a large dynamic range of concentrations without the needfor dilution or other treatment of samples, e.g., over a concentrationrange of more than 10⁵-fold, 10⁶-fold, or 10⁷-fold

The high sensitivity of the instruments, kits, and methods of theinvention allows the establishment of uses for markers, e.g., biologicalmarkers, that have not previously been possible because of a lack ofsensitivity of detection, as well as the establishment of new markers.There are numerous markers currently available which, while potentiallyof use in determining a biological state, are not currently of practicaluse because their lower ranges are unknown. In some cases, abnormallyhigh levels of the marker are detectable by current methodologies, butnormal ranges have not been established. In some cases, upper normalranges of the marker are detectable, but not lower normal ranges, orlevels below normal. In some cases, for example, markers specific totumors, or markers of infection, any level of the marker indicates thepotential presence of the biological state, and enhancing sensitivity ofdetection is an advantage for early diagnosis. In some cases, the rateof change, or lack of change, in the concentration of the marker overmultiple timepoints provides the most useful information, but presentmethods of analysis do not permit timepoint sampling in the early stagesof a condition, when it is typically at its most treatable. In somecases, the marker may be detected at clinically useful levels onlythrough the use of cumbersome methods that are not practical or usefulin a clinical setting, such as methods that require complex sampletreatment and time-consuming analysis. In addition, there are potentialmarkers of biological states that exist in sufficiently lowconcentrations that their presence remains extremely difficult orimpossible to detect by current methods.

The analytical methods and compositions of the present invention providelevels of sensitivity, precision, and robustness that allow thedetection of markers for biological states at concentrations at whichthe markers have been previously undetectable, thus allowing the“repurposing” of such markers from confirmatory markers, or markersuseful only in limited research settings, to diagnostic, prognostic,treatment-directing, or other types of markers useful in clinicalsettings and/or in large-scale clinical settings such as clinicaltrials. Such methods allow the determination of normal and abnormalranges for such markers

The markers thus repurposed can be used for, e.g., detection of normalstate (normal ranges), detection of responder/non-responder (e.g., to atreatment, such as administration of a drug); early disease orpathological occurrence detection (e.g., detection of cancer in itsearliest stages, early detection of cardiac ischemia); disease staging(e.g., cancer); disease monitoring (e.g., diabetes monitoring,monitoring for recurrence of cancer after treatment); study of diseasemechanism; and study of treatment toxicity, such as toxicity of drugtreatments.

The invention thus provides methods and compositions for the sensitivedetection of markers, and further methods of establishing values fornormal and abnormal levels of the markers. In further embodiments, theinvention provides methods of diagnosis, prognosis, and/or treatmentselection based on values established for the markers. The inventionalso provides compositions for use in such methods, e.g., detectionreagents for the ultrasensitive detection of markers.

II. Molecules for Sensitive Detection By the Methods and Compositions ofthe Invention

The instruments, kits and methods of the invention can be used for thesensitive detection and determination of concentration of a number ofdifferent types of single molecules. In particular, the instruments,kits, and methods are useful in the sensitive detection anddetermination of concentration of markers of biological states.“Detection of a single molecule,” as that term is used herein, refers toboth direct and indirect detection. For example, a single molecule maybe labeled with a fluorescent label, and the molecule-label complexdetected in the instruments described herein. Alternatively, a singlemolecule may be labeled with a fluorescent label, then the fluorescentlabel is detached from the single molecule, and the label detected inthe instruments described herein. The term detection of a singlemolecule encompasses both forms of detection.

A. General

Examples of molecules which can be detected using the analyzer andrelated methods of the present invention include: biopolymers such asproteins, nucleic acids, carbohydrates, and small molecules, bothorganic and inorganic. In particular, the instruments, kits, and methodsdescribed herein are useful in the detection of single molecules ofproteins and small molecules in biological samples, and thedetermination of concentration of such molecules in the sample.

The terms “protein,” “polypeptide,” “peptide,” and “oligopeptide,” areused interchangeably herein and include any composition that includestwo or more amino acids joined together by a peptide bond. It may beappreciated that polypeptides can contain amino acids other than the 20amino acids commonly referred to as the 20 naturally occurring aminoacids. Also, polypeptides can include one or more amino acids, includingthe terminal amino acids, which are modified by any means known in theart (whether naturally or non-naturally). Examples of polypeptidemodifications include e.g., by glycosylation, orother-post-translational modification. Modifications which may bepresent in polypeptides of the present invention, include, but are notlimited to, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a polynucleotide or polynucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

The molecules detected by the present instruments, kits, and methods maybe free or may be part of a complex, e.g., an antibody-antigen complex,or more generally a protein-protein complex, e.g., complexes oftroponin.

B. Markers of Biological States

In some embodiments, the invention provides compositions and methods forthe sensitive detection of biological markers, and for the use of suchmarkers in diagnosis, prognosis, and/or determination of methods oftreatment.

Markers of the present invention may be, for example, any compositionand/or molecule or a complex of compositions and/or molecules that isassociated with a biological state of an organism (e.g., a conditionsuch as a disease or a non-disease state). A marker can be, for example,a small molecule, a polypeptide, a nucleic acid, such as DNA and RNA, alipid, such as a phospholipid or a micelle, a cellular component such asa mitochondrion or chloroplast, etc. Markers contemplated by the presentinvention can be previously known or unknown. For example, in someembodiments, the methods herein may identify novel polypeptides that canbe used as markers for a biological state of interest or condition ofinterest, while in other embodiments, known polypeptides are identifiedas markers for a biological state of interest or condition. Using thesystems of the invention it is possible that one can observe thosemarkers, e.g., polypeptides with high potential use in determining thebiological state of an organism, but that are only present at lowconcentrations, such as those “leaked” from diseased tissue. Other highpotentially useful markers or polypeptides may be those that are relatedto the disease, for instance, those that are generated in the tumor-hostenvironment. Any suitable marker that provides information regarding abiological state may be used in the methods and compositions of theinvention. A “marker,” as that term is used herein, encompasses anymolecule that may be detected in a sample from an organism and whosedetection or quantitation provides information about the biologicalstate of the organism.

Biological states include but are not limited to phenotypic states;conditions affecting an organism; states of development; age; health;pathology; disease detection, process, or staging; infection; toxicity;or response to chemical, environmental, or drug factors (such as drugresponse phenotyping, drug toxicity phenotyping, or drug effectivenessphenotyping).

The term “organism” as used herein refers to any living being comprisedof a least one cell. An organism can be as simple as a one cell organismor as complex as a mammal. An organism of the present invention ispreferably a mammal. Such mammal can be, for example, a human or ananimal such as a primate (e.g., a monkey, chimpanzee, etc.), adomesticated animal (e.g., a dog, cat, horse, etc.), farm animal (e.g.,goat, sheep, pig, cattle, etc.), or laboratory animal (e.g., mouse, rat,etc.). Preferably, an organism is a human.

In some embodiments, the methods and compositions of the invention aredirected to classes of markers, e.g., cytokines, growth factors,oncology markers, markers of inflammation, endocrine markers, autoimmunemarkers, thyroid markers, cardiovascular markers, markers of diabetes,markers of infectious disease, neurological markers, respiratorymarkers, gastrointestinal markers, musculoskeletal markers,dermatological disorders, and metabolic markers.

Table 1, below, provides examples of these classes of markers that havebeen measured by the methods and compositions of the invention, andprovides the concentration of the markers as detected by the methods andcompositions of the invention and number of particles that are countedby the single particle analyzer system of the invention for theparticular marker.

TABLE 1 CLASSES OF MARKERS AND EXEMPLARY MARKERS IN THE CLASSES MolarConc. Molecules Cytokines IL-12 p70 2.02 × 10⁻¹⁴ 6.09 × 10⁺⁵ IL-10 5.36× 10⁻¹⁴ 1.61 × 10⁺⁶ IL-1 alpha 5.56 × 10⁻¹⁴ 1.67 × 10⁺⁶ IL-3 5.85 ×10⁻¹⁴ 1.76 × 10⁺⁶ IL-12 p40 6.07 × 10⁻¹⁴ 1.83 × 10⁺⁶ IL-1ra 6.12 × 10⁻¹⁴1.84 × 10⁺⁶ IL-12 8.08 × 10⁻¹⁴ 2.44 × 10⁺⁶ IL-6 9.53 × 10⁻¹⁴ 2.87 × 10⁺⁶IL-4 1.15 × 10⁻¹³ 3.47 × 10⁺⁶ IL-18 1.80 × 10⁻¹³ 5.43 × 10⁺⁶ IP-10 1.88× 10⁻¹³ 1.13 × 10⁺⁷ IL-5 1.99 × 10⁻¹³ 5.98 × 10⁺⁶ Eotaxin 2.06 × 10⁻¹³1.24 × 10⁺⁷ IL-16 3.77 × 10⁻¹³ 1.14 × 10⁺⁷ MIG 3.83 × 10⁻¹³ 1.15 × 10⁺⁷IL-8 4.56 × 10⁻¹³ 1.37 × 10⁺⁷ IL-17 5.18 × 10⁻¹³ 1.56 × 10⁺⁷ IL-7 5.97 ×10⁻¹³ 1.80 × 10⁺⁷ IL-15 6.13 × 10⁻¹³ 1.84 × 10⁺⁷ IL-13 8.46 × 10⁻¹³ 2.55× 10⁺⁷ IL-2R (soluble) 8.89 × 10⁻¹³ 2.68 × 10⁺⁷ IL-2 8.94 × 10⁻¹³ 2.69 ×10⁺⁷ LIF/HILDA 9.09 × 10⁻¹³ 5.47 × 10⁺⁷ IL-1 beta 1.17 × 10⁻¹² 3.51 ×10⁺⁷ Fas/CD95/Apo-1 1.53 × 10⁻¹² 9.24 × 10⁺⁷ MCP-1 2.30 × 10⁻¹² 6.92 ×10⁺⁷ Oncology EGF 4.75 × 10⁻¹⁴ 2.86 × 10⁺⁶ TNF-alpha 6.64 × 10⁻¹⁴ 8.00 ×10⁺⁶ PSA (3rd generation) 1.15 × 10⁻¹³ 6.92 × 10⁺⁶ VEGF 2.31 × 10⁻¹³6.97 × 10⁺⁶ TGF-beta1 2.42 × 10⁻¹³ 3.65 × 10⁺⁷ FGFb 2.81 × 10⁻¹³ 1.69 ×10⁺⁷ TRAIL 5.93 × 10⁻¹³ 3.57 × 10⁺⁷ TNF-RI (p55) 2.17 × 10⁻¹² 2.62 ×10⁺⁸ Inflammation ICAM-1 (soluble) 8.67 × 10⁻¹⁵ 5.22 × 10⁺⁴ RANTES 6.16× 10⁻¹⁴ 3.71 × 10⁺⁶ MIP-2 9.92 × 10⁻¹⁴ 2.99 × 10⁺⁶ MIP-1 beta 1.98 ×10⁻¹³ 5.97 × 10⁺⁶ MIP-1 alpha 2.01 × 10⁻¹³ 6.05 × 10⁺⁶ MMP-3 1.75 ×10⁻¹² 5.28 × 10⁺⁷ Endocrinology 17 beta-Estradiol (E2) 4.69 × 10⁻¹⁴ 2.82× 10⁺⁶ DHEA 4.44 × 10⁻¹³ 2.67 × 10⁺⁷ ACTH 1.32 × 10⁻¹² 7.96 × 10⁺⁷Gastrin 2.19 × 10⁻¹² 1.32 × 10⁺⁸ Growth Hormone (hGH) 2.74 × 10⁻¹² 1.65× 10⁺⁸ Autoimmune GM-CSF 1.35 × 10⁻¹³ 8.15 × 10⁺⁶ C-Reactive Protein(CRP) 3.98 × 10⁻¹³ 2.40 × 10⁺⁷ G-CSF 1.76 × 10⁻¹² 1.06 × 10⁺⁸ ThyroidCyclic AMP 9.02 × 10⁻¹⁵ 5.43 × 10⁺⁵ Calcitonin 3.25 × 10⁻¹⁴ 1.95 × 10⁺⁶Parathyroid Hormone (PTH) 1.56 × 10⁻¹³ 9.37 × 10⁺⁶ CardiovascularB-Natriuretic Peptide 2.86 × 10⁻¹³ 1.72 × 10⁺⁷ NT-proBNP 2.86 × 10⁻¹²8.60 × 10⁺⁷ C-Reactive Protein, HS 3.98 × 10⁻¹³ 2.40 × 10⁺⁷Beta-Thromboglobulin (BTG) 5.59 × 10⁻¹³ 3.36 × 10⁺⁷ Diabetes C-Peptide2.41 × 10⁻¹⁵ 1.45 × 10⁺⁵ Leptin 1.89 × 10⁻¹³ 1.14 × 10⁺⁷ Infectious Dis.IFN-gamma 2.08 × 10⁻¹³ 1.25 × 10⁺⁷ IFN-alpha 4.55 × 10⁻¹³ 2.74 × 10⁺⁷Metabolism Bio-Intact PTH (1-84) 1.59 × 10⁻¹² 1.44 × 10⁺⁸ PTH 1.05 ×10⁻¹³ 9.51 × 10⁺⁶

Cytokines

For both research and diagnostics, cytokines are useful as markers of anumber of conditions, diseases, pathologies, and the like, and thecompositions and methods of the invention include labels for detectionand quantitation of cytokines and methods using such labels to determinenormal and abnormal levels of cytokines, as well as methods ofdiagnosis, prognosis, and/or determination of treatment based on suchlevels.

There are currently over 100 cytokines/chemokines whose coordinate ordiscordant regulation is of clinical interest. In order to correlate aspecific disease process with changes in cytokine levels, the idealapproach requires analyzing a sample for a given cytokine, or multiplecytokines, with high sensitivity. Exemplary cytokines that are presentlyused in marker panels and that may be used in methods and compositionsof the invention include, but are not limited to, BDNF, CREB pS133, CREBTotal, DR-5, EGF, ENA-78, Eotaxin, Fatty Acid Binding Protein,FGF-basic, granulocyte colony-stimulating factor (G-CSF), GCP-2,Granulocyte-macrophage Colony-stimulating Factor GM-CSF (GM-CSF),growth-related oncogene-keratinocytes (GRO-KC), HGF, ICAM-1, IFN-alpha,IFN-gamma, the interleukins IL-10, IL-11, IL-12, IL-12 p40, IL-12p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1alpha,IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, interferon-inducible protein (10 IP-10), JE/MCP-1,keratinocytes (KC), KC/GROa, LIF, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1 (MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation, normal T cell.expressed and secreted (RANTES), Rb (pT821), Rb (total), Rb pSpT249/252,Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumor necrosisfactor-alpha (TNF-alpha), TNF-beta, TNF-RI, TNF-RII, VCAM-1, and VEGF.In some embodiments, the cytokine is IL-12p70, IL-10, IL-1 alpha, IL-3,IL-12 p40, IL-1ra, IL-12, IL-6, IL-4, IL-18, IL-10, IL-5, eotaxin,IL-16, MIG, IL-8, IL-17, IL-7, IL-15, IL-13, IL-2R (soluble), IL-2,LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1, or MCP-1.

Growth Factors

Growth factors include EGF Ligands such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGFR/ErbB Receptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Familysuch as FGF Ligands IGF acidic, FGF-12, FGF basic, FGF-13, FGF-3,FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21,FGF-9, FGF-22, FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGFR1-4, FGF R3, FGF R1, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; theHedgehog Family Desert Hedgehog, Sonic Hedgehog, Indian Hedgehog;Hedgehog Related Molecules & Regulators BOC, GLI-3, CDO, GSK-3alpha/beta, DISP1, GSK-3 alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; theIGF Family IGF Ligands IGF-I, IGF-II, IGF-I Receptor (CD221)IGF-I R, andIGF Binding Protein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6,Cyr61/CCN1, IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10,IGFBP-2, NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor TyrosineKinases Ax1, FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk,IGF-I R, EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2,M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha,EphA7, PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1,EphB2, RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4,Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4VEGF R, FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGFR3/Flt-4; Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan,Agrin, NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators ArylsulfataseA/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS, Exostosin-like 2/EXTL2,HS6ST2, Exostosin-like 3/EXTL3, Iduronate 2-Sulfatase/IDS, GalNAc4S-6ST;SCF, Flt-3 Ligand & M-CSF Flt-3, M-CSF R, Flt-3 Ligand, SCF, M-CSF, SCFR/c-kit; TGF-beta Superfamily (same as listed for inflammatory markers);VEGF/PDGF Family Neuropilin-1, PlGF, Neuropilin-2, PlGF-2, PDGF, VEGF,PDGF R alpha, VEGF-B, PDGF R beta, VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGFR, PDGF-B, VEGF R1/Flt-1, PDGF-C, VEGF R2/KDR/Flk-1, PDGF-D, VEGFR3/Flt-4; Wnt-related Molecules Dickkopf Proteins & Wnt InhibitorsDkk-1,Dkk-4, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled & Related ProteinsFrizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1,Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7,MFRP Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b, Wnt-3a, Wnt-9a, Wnt-4,Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a, Wnt-11, Wnt-7b; OtherWnt-related Molecules APC, Kremen-2, Axin-1, LRP-1, beta-Catenin, LRP-6,Dishevelled-1, Norrin, Dishevelled-3, PKC beta 1, Glypican 3, Pygopus-1,Glypican 5, Pygopus-2, GSK-3 alpha/beta, R-Spondin 1, GSK-3 alpha,R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT, RTK-like Orphan Receptor1/ROR1, Kremen-1, RTK-like Orphan Receptor 2/ROR, and Other GrowthFactors CTGF/CCN2, beta-NGF, Cyr61/CCN1, Norrin, DANCE, NOV/CCN3,EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF, Progranulin, LECT2,Thrombopoietin, LEDGF, WISP-1/CCN4.

Markers of Inflammation

Markers of inflammation include ICAM-1, RANTES, MIP-2, MIP-1-beta,MIP-1-alpha, and MMP-3. Further markers of inflammation include Adhesionmolecules such as the integrins α1β1, α2β1, α3β1, α4β1, α5β1, α6β1,α7β1, α8β1, α9β1, αVβ1, α4β7, α6β4, αDβ2, αLβ2, αMβ2, αVβ3, αVβ5, αVβ6,αVβ8, αXβ2, αIIbβ3, □IELbβ7, beta-2 integrin, beta-3 integrin, beta-2integrin, beta-4 integrin, beta-5 integrin, beta-6 integrin, beta-7integrin, beta-8 integrin, alpha-1 integrin, alpha-2 integrin, alpha-3integrin, alpha-4 integrin, alpha-5 integrin, alpha-6 integrin, alpha-7integrin, alpha-8 integrin, alpha-9 integrin, alpha-D integrin, alpha-Lintegrin, alpha-M integrin, alpha-V integrin, alpha-X integrin,alpha-IIb integrin, alphaIELb integrin; Integrin-associated Moleculessuch as Beta IG-H3, Melusin, CD47, MEPE, CD151, Osteopontin,IBSP/Sialoprotein II, RAGE, IGSF8; Selectins such as E-Selectin,P-Selectin, L-Selectin; Ligands such as CD34, GlyCAM-1, MadCAM-1,PSGL-1, vitronectic, vitronectin receptor, fibronectin, vitronectin,collagen, laminin. ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, PECAM.Further markers of inflammation include Cytokines such as IFN-α, IFN-β,IFN-ε, -κ, -τ, and -ξ, IFN-ω, IFN-γ, IL29, IL28A and IL28B, IL-1, IL-1 □and .□, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,TCCR/WSX-1. Further markers of inflammation include cytokine receptorssuch as Common beta chain, IL-3 R alpha, IL-3 R beta, GM-CSF R, IL-5 Ralpha, Common gamma Chain/IL-2 R gamma, IL-2 R alpha, IL-9 R, IL-2 Rbeta, IL-4 R, IL-21 R, IL-15 R alpha, IL-7 R alpha/CD127, IL-1ra/IL-1F3,IL-1 R8, IL-1 RI, IL-1 R9, IL-1 RII, IL-18 R alpha/IL-1 R5, IL-1 R3/IL-1R AcP, IL-18 R beta/IL-1 R7, IL-1 R4/ST2 SIGIRR, IL-1 R6/IL-1 R rp2,IL-11 R alpha, IL-31 RA, CNTF R alpha, Leptin R, G-CSF R, LIF R alpha,IL-6 R, OSM R beta, IFN-alpha/beta R1, IFN-alpha/beta R2, IFN-gamma R1,IFN-gamma R2, IL-10 R alpha, IL-10 R beta, IL-20 R alpha, IL-20 R beta,IL-22 R, IL-17 R, IL-17 RD, IL-17 RC, IL-17B R, IL-13 R alpha 2, IL-23R, IL-12 R beta 1, IL-12 R beta 2, TCCR/WSX-1, IL-13 R alpha 1. Furthermarkers of inflammation include Chemokines such as CCL-1, CCL-2, CCL-3,CCL-4, CCL-5, CCL-6, CCL-7, CCL-8, CCL-9, CCL-10, CCL-11, CCL-12,CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-20, CCL-21,CCL-22, CCL-23, CCL-24, CCL-25, CCL-26, CCL-27, CCL-28, MCK-2, MIP-2,CINC-1, CINC-2, KC, CINC-3, LIX, GRO, Thymus Chemokine-1, CXCL-1,CXCL-2, CXCL-3, CXCL-4, CXCL-5, CXCL-6, CXCL-7, CXCL-8, CXCL-9, CXCL-10,CXCL-11, CXCL-12, CXCL-13, CXCL-14, CXCL-15, CXCL-16, CXCL-17, XCL1,XCL2, Chemerin. Further markers of inflammation include Chemokinereceptors such as CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7,CCR-8, CCR-9, CCR-10, CXCR3, CXCR6, CXCR4, CXCR1, CXCR5, CXCR2, ChemR23. Further markers of inflammation include Tumor necrosis factors(TNFs), such as TNF.alpha., 4-1BB Ligand/TNFSF9, LIGHT/TNFSF14,APRIL/TNFSF13, Lymphotoxin, BAFF/TNFSF13B, Lymphotoxin beta/TNFSF3, CD27Ligand/TNFSF7, OX40 Ligand/TNFSF4, CD30 Ligand/TNFSF8, TL1A/TNFSF15,CD40 Ligand/TNFSF5, TNF-alpha/TNFSF1A, EDA, TNF-beta/TNFSF1B, EDA-A2,TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18,TWEAK/TNFSF12. Further markers of inflammation include TNF SuperfamilyReceptors such as 4-1BB/TNFRSF9, NGFR/TNFRSF16, BAFF R/TNFRSF13C,Osteoprotegerin/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7,RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5,TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNFRII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A, DR3/TNFRSF25,TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAKR/TNFRSF12, HVEM/TNFRSF14, XEDAR. Further markers of inflammationinclude TNF Superfamily Regulators such as FADD, TRAF-2, RIP1, TRAF-3,TRADD, TRAF-4, TRAF-1, TRAF-6. Further markers of inflammation includeAcute-phase reactants and acute phase proteins. Further markers ofinflammation include TGF-beta superfamily ligands such as Activins,Activin A, Activin B, Activin AB, Activin C, BMPs (Bone MorphogeneticProteins), BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4,BMP-10, BMP-5, BMP-15/GDF-9B, BMP-6, Decapentaplegic,Growth/Differentiation Factors (GDFs), GDF-1, GDF-8, GDF-3, GDF-9 GDF-5,GDF-11, GDF-6, GDF-15, GDF-7, GDNF Family Ligands, Artemin, Neurturin,GDNF, Persephin, TGF-beta, TGF-beta, TGF-beta 3, TGF-beta 1, TGF-beta 5,LAP (TGF-beta 1), Latent TGF-beta bp1, Latent TGF-beta 1, LatentTGF-beta bp2, TGF-beta 1.2, Latent TGF-beta bp4, TGF-beta 2, Lefty,MIS/AMH, Lefty-1, Nodal, Lefty-A, Activin RIA/ALK-2, GFR alpha-1/GDNF Ralpha-1, Activin RIB/ALK-4, GFR alpha-2/GDNF R alpha-2, Activin RIIA,GFR alpha-3/GDNF R alpha-3, Activin RIIB, GFR alpha-4/GDNF R alpha-4,ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-beta RI/ALK-5,BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta RIIb, Endoglin/CD105,TGF-beta RIII. Further markers of inflammation include TGF-betasuperfamily Modulators such as Amnionless, NCAM-1/CD56, BAMBI/NMA,Noggin, BMP-1/PCP, NOMO, Caronte, PRDC, Cerberus 1, SKI, Chordin, Smad1,Chordin-Like 1, Smad2, Chordin-Like 2, Smad3, COCO, Smad4, CRIM1, Smad5,Cripto, Smad7, Crossveinless-2, Smad8, Cryptic, SOST, DAN, LatentTGF-beta bp1, Decorin, Latent TGF-beta bp2, FLRG, Latent TGF-beta bp4,Follistatin, TMEFF1/Tomoregulin-1, Follistatin-like 1, TMEFF2,GASP-1/WFIKKNRP, TSG, GASP-2/WFIKKN, TSK, Gremlin, Vasorin. Furthermarkers of inflammation include EGF Ligands such as Amphiregulin, LRIG3,Betacellulin, Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen,TGF-alpha, Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1.Further markers of inflammation include EGF R/ErbB Receptor Family, suchas EGF R, ErbB3, ErbB2, ErbB4. Further markers of inflammation includeFibrinogen. Further markers of inflammation include SAA. Further markersof inflammation include glial markers, such as alpha.1-antitrypsin,C-reactive protein (CRP), .alpha.2-macroglobulin, glial fibrillaryacidic protein (GFAP), Mac-1, F4/80. Further markers of inflammationinclude myeloperoxidase. Further markers of inflammation includeComplement markers such as C3d, C1q, C5, C4d, C4 bp, and C5a-C9. Furthermarkers of inflammation include Major histocompatibility complex (MHC)glycoproteins, such as HLA-DR and HLA-A, D, C. Further markers ofinflammation include Microglial markers, such as CR3 receptor, MHC I,MHC II, CD 31, CD11a, CD11b, CD11c, CD68, CD45RO, CD45RD, CD18, CD59,CR4, CD45, CD64, and CD44. Further markers of inflammation includealpha.2 macroglobulin receptor, Fibroblast growth factor, Fc gamma RI,Fc gamma RII, CD8, LCA (CD45), CD18 ( ) CD59, Apo J, clusterin, type 2plasminogen activator inhibitor, CD44, Macrophage colony stimulatingfactor receptor, MRP14, 27E10, 4-hydroxynonenal-protein conjugates,I.kappa.B, NF.kappa.B, cPLA.sub.2, COX-2, Matrix metalloproteinases,Membrane lipid peroxidation, and ATPase activity. HSPC228, EMP1, CDC42,TLE3, SPRY2, p40BBP, HSPC060 or NAB2, or a down-regulation of HSPA1A,HSPA1B, MAPRE2 and OAS1 expression, TACE/ADAM17, alpha-1-AcidGlycoprotein, Angiopoietin-1, MIF, Angiopoietin-2, CD14, beta-Defensin2, MMP-2, ECF-L/CHI3L3, MMP-7, EGF, MMP-9, EMAP-II, MSP, EN-RAGE, NitricOxide, Endothelin-1, Osteoactivin/GPNMB, FPR1, PDGF, FPRL1, Pentraxin3/TSG-14, FPRL2, Gas6, PLUNC, GM-CSF, RAGE, S100A10, S100A8, S100A9,HIF-1 alpha, Substance P, TFPI, TGF-beta 1, TIMP-1, TIMP-2, TIMP-3,TIMP-4, TLR4, LBP, TREM-1, Leukotriene A4, Hydrolase TSG-6, Lipocalin-1,uPA, M-CSF, and VEGF

Oncology markers include EGF, TNF-alpha, PSA, VEGF, TGF-beta1, FGFb,TRAIL, and TNF-RI (p55)

Markers of endocrine function include 17 beta-estradiol (E2), DHEA,ACTH, gastrin, and growth hormone (hGH).

Marker of autoimmunity include GM-CSF, C-Reactive Protein, and G-CSF.

Markers of thyroid function include cyclicAMP, calcitonin, andparathyroid hormone.

Cardiovascular markers include cardiac troponin I, cardiac troponin T,B-natriuretic peptide, NT-proBNP, C-ractive Protein HS, andbetatrhromboglobulin.

Makers of diabetes include C-peptide and leptin.

Markers of infectious disease include IFN-gamma and IFN-alpha.

Markers of metabolism include Bio-intact PTH (1-84) and PTH.

Markers of Biological States

Markers may indicate the presence of a particular phenotypic state ofinterest. Examples of phenotypic states include, phenotypes resultingfrom an altered environment, drug treatment, genetic manipulations ormutations, injury, change in diet, aging, or any other characteristic(s)of a single organism or a class or subclass of organisms.

In some embodiments, a phenotypic state of interest is a clinicallydiagnosed disease state. Such disease states include, for example,cancer, cardiovascular disease, inflammatory disease, autoimmunedisease, neurological disease, infectious disease and pregnancy relateddisorders. Alternatively, states of health can be detected usingmarkers.

Cancer phenotypes are included in some aspects of the invention.Examples of cancer herein include, but are not limited to: breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, non-small cell lungcarcinoma gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Cardiovascular disease may be included in other applications of theinvention. Examples of cardiovascular disease include, but are notlimited to, congestive heart failure, high blood pressure, arrhythmias,atherosclerosis, cholesterol, Wolff-Parkinson-White Syndrome, long QTsyndrome, angina pectoris, tachycardia, bradycardia, atrialfibrillation, ventricular fibrillation, congestive heart failure,myocardial ischemia, myocardial infarction, cardiac tamponade,myocarditis, pericarditis, arrhythmogenic right ventricular dysplasia,hypertrophic cardiomyopathy, Williams syndrome, heart valve diseases,endocarditis, bacterial, pulmonary atresia, aortic valve stenosis,Raynaud's disease, Raynaud's disease, cholesterol embolism, Wallenbergsyndrome, Hippel-Lindau disease, and telangiectasis.

Inflammatory disease and autoimmune disease may be included in otherembodiments of the invention. Examples of inflammatory disease andautoimmune disease include, but are not limited to, rheumatoidarthritis, non-specific arthritis, inflammatory disease of the larynx,inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimotothyroidism), colitis, Type 1 diabetes, pelvic inflammatory disease,inflammatory disease of the central nervous system, temporal arteritis,polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa,Reiter's syndrome, scleroderma, systemic lupus and erythematosus.

The methods and compositions of the invention can also providelaboratory information about markers of infectious disease includingmarkers of Adenovirus, Bordetella pertussis, Chlamydia pneumoiae,Chlamydia trachomatis, Cholera Toxin, Cholera Toxin β, Campylobacterjejuni, Cytomegalovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-BarrEA, Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus (HBV) Core,Hepatitis B virus (HBV) Envelope, Hepatitis B virus (HBV) Survace (Ay),Hepatitis C virus (HCV) Core, Hepatitis C virus (HCV) NS3, Hepatitis Cvirus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepititis A, Hepititis D,Hepatitis E virus (HEV) orf2 3 KD, Hepatitis E virus (HEV) orf2 6 KD,Hepatitis E virus (HEV) orf3 3 KD, Human immunodeficiency virus (HIV)-1p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiencyvirus (HIV)-1 gp120, Human papilloma virus (HPV), Herpes simplex virusHSV-1/2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG,Human T-cell leukemia virus (HTLV)-1/2, Influenza A, Influenza A H3N2,Influenza B, Leishmania donovani, Lyme disease, Mumps, M. pneumoniae, M.tuberculosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, PolioVirus, Respiratory syncytial virus (RSV), Rubella, Rubeola, StreptolysinO, Tetanus Toxin, T. pallidum 15 kd, T. pallidum p47, T. cruzi,Toxoplasma, and Varicella Zoster.

III. Labels

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof molecules, e.g., of markers.

One skilled in the art will recognize that many strategies can be usedfor labeling target molecules to enable their detection ordiscrimination in a mixture of particles. The labels may be attached byany known means, including methods that utilize non-specific or specificinteractions of label and target. Labels may provide a detectable signalor affect the mobility of the particle in an electric field. Inaddition, labeling can be accomplished directly or through bindingpartners.

In some embodiments, the label comprises a binding partner to themolecule of interest, where the binding partner is attached to afluorescent moiety. The compositions and methods of the invention mayutilize highly fluorescent moieties, e.g., a moiety capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, wherein the laser is focused ona spot not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. Moieties suitable for thecompositions and methods of the invention are described in more detailbelow.

In some embodiments, the invention provides a label for detecting abiological molecule comprising a binding partner for said biologicalmolecule that is attached to a fluorescent moiety, wherein saidfluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the moiety comprises a plurality offluorescent entities, e.g., about 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, or3-5, 3-6, 3-7, 3-8, 3-9, or 3-10 fluorescent entities. In someembodiments, the moiety comprises about 2 to 4 fluorescent entities. Insome embodiments, biological molecule is a protein or a small molecule,In some embodiments. the biological molecule is a protein. Thefluorescent entities may be fluorescent dye molecules. In someembodiments, the fluorescent dye molecules comprise at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. In some embodiments, the dye molecules areAlexFluor molecules selected from the group consisting of AlexaFluor488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700.In some embodiments, the dye molecules are AlexFluor molecules selectedfrom the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor680 or AlexaFluor 700. In some embodiments, the dye molecules areAlexaFluor647 dye molecules. In some embodiments, the dye moleculescomprise a first type and a second type of dye molecules, e.g., twodifferent AlexaFluor molecules, e.g., where the first type and secondtype of dye molecules have different emission spectra. The ratio of thenumber of first type to second type of dye molecule may be, e.g., 4:1,3:1, 2:1, 1:1, 1:2, 1:3 or 1:4. The binding partner may be, e.g., anantibody.

In some embodiments, the invention provides a label for the detection ofa marker, wherein the label comprises a binding partner for the markerand a fluorescent moiety, wherein the fluorescent moiety is capable ofemitting at least about 200 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, wherein the laser isfocused on a spot not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the fluorescent moiety comprises a fluorescent molecule. In someembodiments, the fluorescent moiety comprises a plurality of fluorescentmolecules, e.g., about 2-10, 2-8, 2-6, 2-4, 3-10, 3-8, 3-6 fluorescentmolecules. In some embodiments, the label comprises about 2-4fluorescent molecules. In some embodiments, the fluorescent dyemolecules comprise at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the fluorescent molecules are selected from the groupconsisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor680 or AlexaFluor 700. In some embodiments, the fluorescent moleculesare selected from the group consisting of AlexaFluor 488, AlexaFluor532, AlexaFluor 680 or AlexaFluor 700. In some embodiments, the bindingpartner comprises an antibody. In some embodiments, the antibody is amonoclonal antibody. In some embodiments, antibody is a polyclonalantibody.

The antibody may be specific to any suitable marker. In someembodiments, the antibody is specific to a marker that is selected fromthe group consisting of cytokines, growth factors, oncology markers,markers of inflammation, endocrine markers, autoimmune markers, thyroidmarkers, cardiovascular markers, markers of diabetes, markers ofinfectious disease, neurological markers, respiratory markers,gastrointestinal markers, musculoskeletal markers, dermatologicaldisorders, and metabolic markers.

In some embodiments, the antibody is specific to a marker that is acytokine. In some embodiments, the cytokine is selected from the groupconsisting of BDNF, CREB pS133, CREB Total, DR-5, EGF, ENA-78, Eotaxin,Fatty Acid Binding Protein, FGF-basic, granulocyte colony-stimulatingfactor (G-CSF), GCP-2, Granulocyte-macrophage Colony-stimulating FactorGM-CSF (GM-CSF), growth-related oncogene-keratinocytes (GRO-KC), HGF,ICAM-1, IFN-alpha, IFN-gamma, the interleukins IL-10, IL-11, IL-12,IL-12 p40, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-16, IL-17, IL-18,IL-1alpha, IL-1beta, IL-1ra, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, interferon-inducible protein (10 IP-10),JE/MCP-1, keratinocytes (KC), KC/GROa, LIF, Lymphotacin, M-CSF, monocytechemoattractant protein-1 (MCP-1), MCP-1 (MCAF), MCP-3, MCP-5, MDC, MIG,macrophage inflammatory (MIP-1 alpha), MIP-1 beta, MIP-1 gamma, MIP-2,MIP-3 beta, OSM, PDGF-BB, regulated upon activation, normal T cell.expressed and secreted (RANTES), Rb (pT821), Rb (total), Rb pSpT249/252,Tau (pS214), Tau (pS396), Tau (total), Tissue Factor, tumor necrosisfactor-alpha (TNF-alpha), TNF-beta, TNF-RI, TNF-RII, VCAM-1, and VEGF.

In some embodiments, the cytokine is selected from the group consistingof IL-12p70, IL-10, IL-1 alpha, IL-3, IL-12 p40, IL-1ra, IL-12, IL-6,IL-4, IL-18, IL-10, IL-5, Eotaxin, IL-16, MIG, IL-8, IL-17, IL-7, IL-15,IL-13, IL-2R (soluble), IL-2, LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1 andMCP-1.

In some embodiments, the antibody is specific to a marker that is agrowth factor. In some embodiments, the antibody is specific to a markerthat is a growth factor that is TGF-beta. In some embodiments, thegrowth factor is GF Ligands such as Amphiregulin, LRIG3, Betacellulin,Neuregulin-1/NRG1, EGF, Neuregulin-3/NRG3, Epigen, TGF-alpha,Epiregulin, TMEFF1/Tomoregulin-1, HB-EGF, TMEFF2, LRIG1; EGF R/ErbBReceptor Family such as EGF R, ErbB3, ErbB2, ErbB4; FGF Family such asFGF LigandsIGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4,FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-22,FGF-10, FGF-23, FGF-11, KGF/FGF-7, FGF Receptors FGF R1-4, FGF R3, FGFR1, FGF R4, FGF R2, FGF R5, FGF Regulators FGF-BP; the Hedgehog FamilyDesert Hedgehog, Sonic Hedgehog, Indian Hedgehog; Hedgehog RelatedMolecules & Regulators BOC, GLI-3, CDO, GSK-3 alpha/beta, DISP1, GSK-3alpha, Gas1, GSK-3 beta, GLI-1, Hip, GLI-2; the IGF FamilyIGFLigandsIGF-I, IGF-II, IGF-I Receptor (CD221)IGF-I R, and IGF BindingProtein (IGFBP) Family ALS, IGFBP-5, CTGF/CCN2, IGFBP-6, Cyr61/CCN1,IGFBP-L1, Endocan, IGFBP-rp1/IGFBP-7, IGFBP-1, IGFBP-rP10, IGFBP-2,NOV/CCN3, IGFBP-3, WISP-1/CCN4, IGFBP-4; Receptor Tyrosine Kinases Ax1,FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R,EGF, R IGF-II R, Eph, INSRR, EphA1, Insulin R/CD220, EphA2, M-CSF R,EphA3, Mer, EpbA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7,PDGF R beta, EphA8, Ret, EphB1, RTK-like Orphan Receptor 1/ROR1, EphB2,RTK-like Orphan Receptor 2/ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1,EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF, R1-4 VEGF R,FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/KDR/Flk-1, FGF R3, VEGF R3/Flt-4;Proteoglycans & Regulators Proteoglycans Aggrecan, Mimecan, Agrin,NG2/MCSP, Biglycan, Osteoadherin, Decorin, Podocan, DSPG3,delta-Sarcoglycan, Endocan, Syndecan-1/CD138, Endoglycan, Syndecan-2,Endorepellin/Perlecan, Syndecan-3, Glypican 2, Syndecan-4, Glypican 3,Testican 1/SPOCK1, Glypican 5, Testican 2/SPOCK2, Glypican 6, Testican3/SPOCK3, Lumican, Versican, Proteoglycan Regulators

Arylsulfatase A/ARSA, Glucosamine (N-acetyl)-6-Sulfatase/GNS,Exostosin-like 2/EXTL2, HS6ST2, Exostosin-like 3/EXTL3, Iduronate2-Sulfatase/IDS, GalNAc4S-6ST; SCF, Flt-3 Ligand & M-CSF Flt-3, M-CSF R,Flt-3 Ligand, SCF, M-CSF, SCF R/c-kit; TGF-beta Superfamily (same aslisted for inflammatory markers); VEGF/PDGF Family Neuropilin-1, PlGF,Neuropilin-2, PlGF-2, PDGF, VEGF, PDGF R alpha, VEGF-B, PDGF R beta,VEGF-C, PDGF-A, VEGF-D, PDGF-AB, VEGF R, PDGF-B, VEGF R1/Flt-1, PDGF-C,VEGF R2/KDR/Flk-1, PDGF-D, VEGF R3/Flt-4; Wnt-related Molecules DickkopfProteins & Wnt InhibitorsDkk-1, Dkk-2, Soggy-1, Dkk-3, WIF-1 Frizzled &Related Proteins Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9,Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6,sFRP-4, Frizzled-7, MFRP Wnt Ligands Wnt-1, Wnt-8a, Wnt-2b, Wnt-8b,Wnt-3a, Wnt-9a, Wnt-4, Wnt-9b, Wnt-5a, Wnt-10a, Wnt-5b, Wnt-10b, Wnt-7a,Wnt-11, Wnt-7b; Other Wnt-related Molecules APC, Kremen-2, Axin-1,LRP-1, beta-Catenin, LRP-6, Dishevelled-1, Norrin, Dishevelled-3, PKCbeta 1, Glypican 3, Pygopus-1, Glypican 5, Pygopus-2, GSK-3 alpha/beta,R-Spondin 1, GSK-3 alpha, R-Spondin 2, GSK-3 beta, R-Spondin 3, ICAT,RTK-like Orphan Receptor 1/ROR1, Kremen-1, RTK-like Orphan Receptor2/ROR, and Other Growth Factors CTGF/CCN2, beta-NGF, Cyr61/CCN1, Norrin,DANCE, NOV/CCN3, EG-VEGF/PK1, Osteocrin, Hepassocin, PD-ECGF, HGF,Progranulin, LECT2, Thrombopoietin, LEDGF, or WISP-1/CCN4.

In some embodiments, the antibody is specific to a marker that is amarker for cancer (oncology marker). In some embodiments, the antibodyis specific to a marker that is a marker for cancer that is EGF. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is TNF-alpha. In some embodiments, the antibody is specificto a marker that is a marker for cancer that is PSA. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is VEGF. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TGF-beta. In someembodiments, the antibody is specific to a marker that is a marker forcancer that is FGFb. In some embodiments, the antibody is specific to amarker that is a marker for cancer that is TRAIL. In some embodiments,the antibody is specific to a marker that is a marker for cancer that isTNF-RI (p55).

In further embodiments, the antibody is specific to a marker for cancerthat is alpha-Fetoprotein. In some embodiments, the antibody is specificto a marker for cancer that is ER beta/NR3A2. In some embodiments, theantibody is specific to a marker for cancer that is ErbB2. In someembodiments, the antibody is specific to a marker for cancer that isKallikrein 3/PSA. In some embodiments, the antibody is specific to amarker for cancer that is ER alpha/NR3A1. In some embodiments, theantibody is specific to a marker for cancer that is ProgesteroneR/NR3C3. In some embodiments, the antibody is specific to a marker forcancer that is A33. In some embodiments, the antibody is specific to amarker for cancer that is MIA. In some embodiments, the antibody isspecific to a marker for cancer that is Aurora A. In some embodiments,the antibody is specific to a marker for cancer that is MMP-2. In someembodiments, the antibody is specific to a marker for cancer that isBcl-2. In some embodiments, the antibody is specific to a marker forcancer that is MMP-3. In some embodiments, the antibody is specific to amarker for cancer that is Cadherin-13. In some embodiments, the antibodyis specific to a marker for cancer that is MMP-9. In some embodiments,the antibody is specific to a marker for cancer that is E-Cadherin. Insome embodiments, the antibody is specific to a marker for cancer thatis NEK2. In some embodiments, the antibody is specific to a marker forcancer that is Carbonic Anhydrase IX. In some embodiments, the antibodyis specific to a marker for cancer that is Nestin. In some embodiments,the antibody is specific to a marker for cancer that is beta-Catenin. Insome embodiments, the antibody is specific to a marker for cancer thatis NG2/MCSP. In some embodiments, the antibody is specific to a markerfor cancer that is Cathepsin D. In some embodiments, the antibody isspecific to a marker for cancer that is Osteopontin. In someembodiments, the antibody is specific to a marker for cancer that isCD44. In some embodiments, the antibody is specific to a marker forcancer that is p21/CIP1/CDKN1A. In some embodiments, the antibody isspecific to a marker for cancer that is CEACAM-6. In some embodiments,the antibody is specific to a marker for cancer that is p27/Kip1. Insome embodiments, the antibody is specific to a marker for cancer thatis Cornulin. In some embodiments, the antibody is specific to a markerfor cancer that is p53. In some embodiments, the antibody is specific toa marker for cancer that is DPPA4. In some embodiments, the antibody isspecific to a marker for cancer that is Prolactin. In some embodiments,the antibody is specific to a marker for cancer that is ECM-1. In someembodiments, the antibody is specific to a marker for cancer that isPSP94. In some embodiments, the antibody is specific to a marker forcancer that is EGF. In some embodiments, the antibody is specific to amarker for cancer that is S100B. In some embodiments, the antibody isspecific to a marker for cancer that is EGF R. In some embodiments, theantibody is specific to a marker for cancer that is S100P. In someembodiments, the antibody is specific to a marker for cancer that isEMMPRIN/CD147. In some embodiments, the antibody is specific to a markerfor cancer that is SCF R/c-kit. In some embodiments, the antibody isspecific to a marker for cancer that is Fibroblast Activation Proteinalpha/FAP. In some embodiments, the antibody is specific to a marker forcancer that is Serpin E1/PAI-1. In some embodiments, the antibody isspecific to a marker for cancer that is FGF acidic. In some embodiments,the antibody is specific to a marker for cancer that is Serum AmyloidA4. In some embodiments, the antibody is specific to a marker for cancerthat is FGF basic. In some embodiments, the antibody is specific to amarker for cancer that is Survivin. In some embodiments, the antibody isspecific to a marker for cancer that is Galectin-3. In some embodiments,the antibody is specific to a marker for cancer that is TEM8. In someembodiments, the antibody is specific to a marker for cancer that isGlypican 3. In some embodiments, the antibody is specific to a markerfor cancer that is TIMP-1. In some embodiments, the antibody is specificto a marker for cancer that is HIN-1/Secretoglobulin 3A1. In someembodiments, the antibody is specific to a marker for cancer that isTIMP-2. In some embodiments, the antibody is specific to a marker forcancer that is IGF-I. In some embodiments, the antibody is specific to amarker for cancer that is TIMP-3. In some embodiments, the antibody isspecific to a marker for cancer that is IGFBP-3. In some embodiments,the antibody is specific to a marker for cancer that is TIMP-4. In someembodiments, the antibody is specific to a marker for cancer that isIL-6. In some embodiments, the antibody is specific to a marker forcancer that is TNF-alpha/TNFSF1A. In some embodiments, the antibody isspecific to a marker for cancer that is Kallikrein 6/Neurosin. In someembodiments, the antibody is specific to a marker for cancer that isTRAF-4. In some embodiments, the antibody is specific to a marker forcancer that is M-CSF. In some embodiments, the antibody is specific to amarker for cancer that is uPA. In some embodiments, the antibody isspecific to a marker for cancer that is Matriptase/ST14. In someembodiments, the antibody is specific to a marker for cancer that isuPAR. In some embodiments, the antibody is specific to a marker forcancer that is Mesothelin. In some embodiments, the antibody is specificto a marker for cancer that is VCAM-1. In some embodiments, the antibodyis specific to a marker for cancer that is Methionine Aminopeptidase. Insome embodiments, the antibody is specific to a marker for cancer thatis VEGF. In some embodiments, the antibody is specific to a marker forcancer that is Methionine Aminopeptidase 2.

In some embodiments, the antibody is specific to a marker that is amarker for inflammation. In some embodiments, the antibody is specificto a marker that is a marker for inflammation that is ICAM-1. In someembodiments, the antibody is specific to a marker that is a marker forinflammation that is RANTES. In some embodiments, the antibody isspecific to a marker that is a marker for inflammation that is MIP-2. Insome embodiments, the antibody is specific to a marker that is a markerfor inflammation that is MIP-1 beta. In some embodiments, the antibodyis specific to a marker that is a marker for inflammation that is MIP-1alpha. In some embodiments, the antibody is specific to a marker that isa marker for inflammation that is MMP-3.

In some embodiments, the antibody is specific to a marker that is amarker for endocrine function. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that is 17beta-estradiol (E2). In some embodiments, the antibody is specific to amarker that is a marker for endocrine function that is DHEA. In someembodiments, the antibody is specific to a marker that is a marker forendocrine function that is ACTH. In some embodiments, the antibody isspecific to a marker that is a marker for endocrine function that isgastrin. In some embodiments, the antibody is specific to a marker thatis a marker for endocrine function that is growth hormone.

In some embodiments, the antibody is specific to a marker that is amarker for autoimmune disease. In some embodiments, the antibody isspecific to a marker that is a marker for autoimmune disease that isGM-CSF. In some embodiments, the antibody is specific to a marker thatis a marker for autoimmune disease that is C-reactive protein (CRP). Insome embodiments, the antibody is specific to a marker that is a markerfor autoimmune disease that is G-CSF.

In some embodiments, the antibody is specific to a marker for thyroidfunction. In some embodiments, the antibody is specific to a marker forthyroid function that is cyclic AMP. In some embodiments, the antibodyis specific to a marker for thyroid function. In some embodiments, theantibody is specific to a marker for thyroid function that iscalcitonin. In some embodiments, the antibody is specific to a markerfor thyroid function. In some embodiments, the antibody is specific to amarker for thyroid function that is parathyroid hormone.

In some embodiments, the antibody is specific to a marker forcardiovascular function. In some embodiments, the antibody is specificto a marker for cardiovascular function that is B-natruiretic peptide.In some embodiments, the antibody is specific to a marker forcardiovascular function that is NT-proBNP. In some embodiments, theantibody is specific to a marker for cardiovascular function that isC-reactive protein, HS. In some embodiments, the antibody is specific toa marker for cardiovascular function that is beta-thromboglobulin. Insome embodiments, the antibody is specific to a marker forcardiovascular function that is a cardiac troponin. In some embodiments,the antibody is specific to a marker for cardiovascular function that iscardiac troponin I. In some embodiments, the antibody is specific to amarker for cardiovascular function that is cardiac troponin T.

In some embodiments, the antibody is specific to a marker for diabetes.In some embodiments, the antibody is specific to a marker for diabetesthat is C-peptide. In some embodiments, the antibody is specific to amarker for diabetes that is leptin.

In some embodiments, the antibody is specific to a marker for infectiousdisease. In some embodiments, the antibody is specific to a marker forinfectious disease that is IFN gamma. In some embodiments, the antibodyis specific to a marker for infectious disease that is IFN alpha. Insome embodiments, the antibody is specific to a marker for infectiousdisease that is TREM-1.

In some embodiments, the antibody is specific to a marker formetabolism. In some embodiments, the antibody is specific to a markerfor metabolism that is bio-intact PTH (1-84). In some embodiments, theantibody is specific to a marker for metabolism that is PTH.

In some embodiments, the antibody is specific to a marker that is IL-1beta. In some embodiments, the antibody is specific to a marker that isTNF-alpha. In some embodiments, the antibody is specific to a markerthat is IL-6. In some embodiments, the antibody is specific to a markerthat is TnI (cardiac troponin I). In some embodiments, the antibody isspecific to a marker that is IL-8.

In some embodiments, the antibody is specific to a marker that is Abeta40. In some embodiments, the antibody is specific to a marker that isAbeta 42. In some embodiments, the antibody is specific to a marker thatis cAMP. In some embodiments, the antibody is specific to a marker thatis FAS Ligand. In some embodiments, the antibody is specific to a markerthat is FGF-basic. In some embodiments, the antibody is specific to amarker that is GM-CSF. In some embodiments, the antibody is specific toa marker that is IFN-alpha. In some embodiments, the antibody isspecific to a marker that is IFN-gamma. In some embodiments, theantibody is specific to a marker that is IL-1a. In some embodiments, theantibody is specific to a marker that is IL-2. In some embodiments, theantibody is specific to a marker that is IL-4. In some embodiments, theantibody is specific to a marker that is IL-5. In some embodiments, theantibody is specific to a marker that is IL-7. In some embodiments, theantibody is specific to a marker that is IL-12. In some embodiments, theantibody is specific to a marker that is In some embodiments, theantibody is specific to a marker that is IL-13. In some embodiments, theantibody is specific to a marker that is IL-17. In some embodiments, theantibody is specific to a marker that is MCP-1. In some embodiments, theantibody is specific to a marker that is MIP-1a. In some embodiments,the antibody is specific to a marker that is RANTES. In someembodiments, the antibody is specific to a marker that is VEGF.

In some embodiments, the antibody is specific to a marker that is ACE.In some embodiments, the antibody is specific to a marker that isactivin A. In some embodiments, the antibody is specific to a markerthat is adiponectin. In some embodiments, the antibody is specific to amarker that is adipsin. In some embodiments, the antibody is specific toa marker that is AgRP. In some embodiments, the antibody is specific toa marker that is AKT1. In some embodiments, the antibody is specific toa marker that is albumin. In some embodiments, the antibody is specificto a marker that is betacellulin. In some embodiments, the antibody isspecific to a marker that is bombesin. In some embodiments, the antibodyis specific to a marker that is CD14. In some embodiments, the antibodyis specific to a marker that is CD-26. In some embodiments, the antibodyis specific to a marker that is CD-38. In some embodiments, the antibodyis specific to a marker that is CD-40L. In some embodiments, theantibody is specific to a marker that is CD-40s. In some embodiments,the antibody is specific to a marker that is CDK5. In some embodiments,the antibody is specific to a marker that is Complement C3. In someembodiments, the antibody is specific to a marker that is Complement C4.In some embodiments, the antibody is specific to a marker that isC-peptide. In some embodiments, the antibody is specific to a markerthat is CRP. In some embodiments, the antibody is specific to a markerthat is EGF. In some embodiments, the antibody is specific to a markerthat is E-selectin. In some embodiments, the antibody is specific to amarker that is FAS. In some embodiments, the antibody is specific to amarker that is FASLG. In some embodiments, the antibody is specific to amarker that is Fetuin A. In some embodiments, the antibody is specificto a marker that is fibrinogen. In some embodiments, the antibody isspecific to a marker that is ghrelin. In some embodiments, the antibodyis specific to a marker that is glucagon. In some embodiments, theantibody is specific to a marker that is growth hormone. In someembodiments, the antibody is specific to a marker that is haptoglobulin.In some embodiments, the antibody is specific to a marker that ishepatocyte growth factor. In some embodiments, the antibody is specificto a marker that is HGF. In some embodiments, the antibody is specificto a marker that is ICAM 1. In some embodiments, the antibody isspecific to a marker that is IFNG. In some embodiments, the antibody isspecific to a marker that is IGF1. In some embodiments, the antibody isspecific to a marker that is IL-1RA. In some embodiments, the antibodyis specific to a marker that is Il-6sr. In some embodiments, theantibody is specific to a marker that is IL-8. In some embodiments, theantibody is specific to a marker that is IL-10. In some embodiments, theantibody is specific to a marker that is IL-18. In some embodiments, theantibody is specific to a marker that is ILGFBP1. In some embodiments,the antibody is specific to a marker that is ILGFBP3. In someembodiments, the antibody is specific to a marker that is insulin-likegrowth factor 1. In some embodiments, the antibody is specific to amarker that is LEP. In some embodiments, the antibody is specific to amarker that is M-CSF. In some embodiments, the antibody is specific to amarker that is MMP2. In some embodiments, the antibody is specific to amarker that is MMP9. In some embodiments, the antibody is specific to amarker that is NGF. In some embodiments, the antibody is specific to amarker that is PAI-1. In some embodiments, the antibody is specific to amarker that is RAGE. In some embodiments, the antibody is specific to amarker that is RSP4. In some embodiments, the antibody is specific to amarker that is resistin. In some embodiments, the antibody is specificto a marker that is sex hormone binding globulin. In some embodiments,the antibody is specific to a marker that is SOCX3. In some embodiments,the antibody is specific to a marker that is TGF beta. In someembodiments, the antibody is specific to a marker that isthromboplastin. In some embodiments, the antibody is specific to amarker that is TNF R1. In some embodiments, the antibody is specific toa marker that is VCAM-1. In some embodiments, the antibody is specificto a marker that is VWF. In some embodiments, the antibody is specificto a marker that is TSH. In some embodiments, the antibody is specificto a marker that is EPITOME.

In some embodiments, the antibody is specific to a marker that iscardiac troponin I. In some embodiments, the antibody is specific to amarker that is TREM-1. In some embodiments, the antibody is specific toa marker that is IL-6. In some embodiments, the antibody is specific toa marker that is IL-8. In some embodiments, the antibody is specific toa marker that is Leukotriene T4. In some embodiments, the antibody isspecific to a marker that is Akt1. In some embodiments, the antibody isspecific to a marker that is TGF-beta. In some embodiments, the antibodyis specific to a marker that is Fas ligand.

In some embodiments, the fluorescent moiety comprises a fluorescentmolecule. In some embodiments, the fluorescent moiety comprises aplurality of fluorescent molecules, e.g., about 2-10, 2-8, 2-6, 2-4,3-10, 3-8, or 3-6 fluorescent molecules. In some embodiments, the labelcomprises about 2-4 fluorescent molecules. In some embodiments, thefluorescent molecule comprises a molecule that comprises at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. group. In some embodiments, the fluorescentmolecules are selected from the group consisting of AlexaFluor 488,AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. Insome embodiments, the fluorescent molecules are selected from the groupconsisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 680 orAlexaFluor 700. In some embodiments, the fluorescent molecules areAlexaFluor 647 molecules.

A. Binding Partners

Any suitable binding partner with the requisite specificity for the formof molecule, e.g., marker, to be detected may be used. If the molecule,e.g., marker, has several different forms, various specificities ofbinding partners are possible. Suitable binding partners are known inthe art and include antibodies, aptamers, lectins, and receptors. Auseful and versatile type of binding partner is an antibody.

1. Antibodies

Thus, in some embodiments, the binding partner is an antibody specificfor a molecule to be detected. The term “antibody,” as used herein, is abroad term and is used in its ordinary sense, including, withoutlimitation, to refer to naturally occurring antibodies as well asnon-naturally occurring antibodies, including, for example, single chainantibodies, chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof. It will be appreciated that thechoice of epitope or region of the molecule to which the antibody israised will determine its specificity, e.g., for various forms of themolecule, if present, or for total (e.g., all, or substantially all ofthe molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc.,Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA;Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049USA; BiosPacific, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Capture binding partners and detection binding partner pairs, e.g.,capture and detection antibody pairs, may be used in embodiments of theinvention. Thus, in some embodiments, a heterogeneous assay protocol isused in which, typically, two binding partners, e.g., two antibodies,are used. One binding partner is a capture partner, usually immobilizedon a solid support, and the other binding partner is a detection bindingpartner, typically with a detectable label attached. Such antibody pairsare available from the sources described above, e.g., BiosPacific,Emeryville, Calif. Antibody pairs can also be designed and prepared bymethods well-known in the art. Compositions of the invention includeantibody pairs wherein one member of the antibody pair is a label asdescribed herein, and the other member is a capture antibody.

In some embodiments it is useful to use an antibody that cross-reactswith a variety of species, either as a capture antibody, a detectionantibody, or both. Such embodiments include the measurement of drugtoxicity by determining, e.g., release of cardiac troponin into theblood as a marker of cardiac damage. A cross-reacting antibody allowsstudies of toxicity to be done in one species, e.g. a non-human species,and direct transfer of the results to studies or clinical observationsof another species, e.g., humans, using the same antibody or antibodypair in the reagents of the assays, thus decreasing variability betweenassays. Thus, in some embodiments, one or more of the antibodies for useas a binding partner to the marker, e.g., cardiac troponin, such ascardiac troponin I, may be a cross-reacting antibody. In someembodiments, the antibody cross-reacts with the marker, e.g. cardiactroponin, from at least two species selected from the group consistingof human, monkey, dog, and mouse. In some embodiments the antibodycross-reacts with the marker e.g. cardiac troponin, from all of thegroup consisting of human, monkey, dog, and mouse.

B. Fluorescent Moieties

In some embodiments of labels used in the invention, the bindingpartner, e.g., antibody, is attached to a fluorescent moiety. Thefluorescence of the moiety will be sufficient to allow detection in asingle molecule detector, such as the single molecule detectorsdescribed herein.

A “fluorescent moiety,” as that term is used herein, includes one ormore fluorescent entities whose total fluorescence is such that themoiety may be detected in the single molecule detectors describedherein. Thus, a fluorescent moiety may comprise a single entity (e.g., aQuantum Dot or fluorescent molecule) or a plurality of entities (e.g., aplurality of fluorescent molecules). It will be appreciated that when“moiety,” as that term is used herein, refers to a group of fluorescententities, e.g., a plurality of fluorescent dye molecules, eachindividual entity may be attached to the binding partner separately orthe entities may be attached together, as long as the entities as agroup provide sufficient fluorescence to be detected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired limit of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of a molecule, e.g., a marker, at a limitof detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001,0.00001, or 0.000001 pg/ml and with a coefficient of variation of lessthan about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% orless, e.g., about 10% or less, in the instruments described herein. Insome embodiments, the fluorescence of the fluorescent moiety is suchthat it allows detection and/or quantitation of a molecule, e.g., amarker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of lessthan about 10%, in the instruments described herein.

“Limit of detection,” as that term is used herein, includes the lowestconcentration at which one can identify a sample as containing amolecule of the substance of interest, e.g., the first non-zero value.It can be defined by the variability of zeros and the slope of thestandard curve. For example, the limit of detection of an assay may bedetermined by running a standard curve, determining the standard curvezero value, and adding 2 standard deviations to that value. Aconcentration of the substance of interest that produces a signal equalto this value is the “lower limit of detection” concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties that are preferred arefluorescent moieties, e.g., dye molecules that have a combination of 1)high absorption coefficient; 2) high quantum yield; 3) highphotostability (low photobleaching); and 4) compatibility with labelingthe molecule of interest (e.g., protein) so that it may be analyzedusing the analyzers and systems of the invention (e.g., does not causeprecipitation of the protein of interest, or precipitation of a proteinto which the moiety has been attached).

Fluorescent moieties, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that are useful in someembodiments of the invention may be defined in terms of their photonemission characteristics when stimulated by EM radiation. For example,in some embodiments, the invention utilizes a fluorescent moiety, e.g.,a moiety comprising a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000, photons whensimulated by a laser emitting light at the excitation wavelength of themoiety, where the laser is focused on a spot of not less than about 0.5microns in diameter that contains the moiety, and where the total energydirected at the spot by the laser is no more than about 3 microJoules.It will be appreciated that the total energy may be achieved by manydifferent combinations of power output of the laser and length of timeof exposure of the dye moiety. E.g., a laser of a power output of 1 mWmay be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms,and so on.

In some embodiments, the invention utilizes a fluorescent dye moiety,e.g., a single fluorescent dye molecule or a plurality of fluorescentdye molecules, that is capable of emitting an average of at least about50 photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye moiety, e.g., a single fluorescent dye molecule or aplurality of fluorescent dye molecules, that is capable of emitting anaverage of at least about 100 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye moiety, e.g., a singlefluorescent dye molecule or a plurality of fluorescent dye molecules,that is capable of emitting an average of at least about 150 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, where the laser is focused on a spot of not less than about5 microns in diameter that contains the moiety, and wherein the totalenergy directed, at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye moiety, e.g., a single fluorescent dye molecule or a plurality offluorescent dye molecules, that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. In some embodiments, the inventionutilizes a fluorescent dye moiety, e.g., a single fluorescent dyemolecule or a plurality of fluorescent dye molecules, that is capable ofemitting an average of at least about 300 photons when simulated by alaser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. In someembodiments, the invention utilizes a fluorescent dye moiety e.g., asingle fluorescent dye molecule or a plurality of fluorescent dyemolecules, that is capable of emitting an average of at least about 500photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the moiety, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules.

In some embodiments, the fluorescent moiety comprises an average of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, e.g.,fluorescent molecules. In some embodiments, the fluorescent moietycomprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 fluorescent entities, e.g., fluorescent molecules. In someembodiments, the fluorescent moiety comprises an average of about 1 to11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5,or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 2 to 8fluorescent moieties are attached. In some embodiments, an average ofabout 2 to 6 fluorescent entities. In some embodiments, the fluorescentmoiety comprises an average of about 2 to 4 fluorescent entities. Insome embodiments, the fluorescent moiety comprises an average of about 3to 10 fluorescent entities. In some embodiments, the fluorescent moietycomprises an average of about 3 to 8 fluorescent entities. In someembodiments, the fluorescent moiety comprises an average of about 3 to 6fluorescent entities. By “average” is meant that, in a given sample thatis a representative sample of a group of labels of the invention, wherethe sample contains a plurality of the binding partner-fluorescentmoiety units, the molar ratio of the particular fluorescent entity ofwhich the fluorescent moiety is comprise, to the binding partner, asdetermined by standard analytical methods, corresponds to the number orrange of numbers specified.For example, in embodiments in which thelabel comprises a binding partner that is an antibody and a fluorescentmoiety that comprises a plurality of fluorescent dye molecules of aspecific absorbance, a spectrophometric assay may be used in which asolution of the label is diluted to an appropriate level and theabsorbance at 280 nm is taken to determine the molarity of the protein(antibody) and an absorbance at, e.g., 650 nm (for AlexaFluor 647) istaken to determine the molarity of the fluorescent dye molecule. Theratio of the latter molarity to the former represents the average numberof fluorescent entities (dye molecules) in the fluorescent moietyattached to each antibody.

1. Dyes

In some embodiments, the invention utilizes fluorescent moieties thatcomprise fluorescent dye molecules. In some embodiments, the inventionutilizes a fluorescent dye molecule that is capable of emitting anaverage of at least about 50 photons when simulated by a laser emittinglight at the excitation wavelength of the molecule, where the laser isfocused on a spot of not less than about 5 microns in diameter thatcontains the molecule, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the invention utilizes a fluorescent dye molecule that is capable ofemitting an average of at least about 75 photons when simulated by alaser emitting light at the excitation wavelength of the molecule, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the molecule, and wherein the total energydirected at the spot by the laser is no more than about 3 microJoules.In some embodiments, the invention utilizes a fluorescent dye moleculethat is capable of emitting an average of at least about 100 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe molecule, where the laser is focused on a spot of not less thanabout 5 microns in diameter that contains the molecule, and wherein thetotal energy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the invention utilizes a fluorescentdye molecule that is capable of emitting an average of at least about150 photons when simulated by a laser emitting light at the excitationwavelength of the molecule, where the laser is focused on a spot of notless than about 5 microns in diameter that contains the molecule, andwherein the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the invention utilizes afluorescent dye molecule that is capable of emitting an average of atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the molecule, where the laser is focused on aspot of not less than about 5 microns in diameter that contains themolecule, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules

A non-inclusive list of useful fluorescent entities for use in thefluorescent moieties of the invention is given in Table 2, below. Insome embodiments, the fluorescent dye is selected from the groupconsisting of Alexa Flour 488, 532, 647, 700, 750, Fluorescein,B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605. In someembodiments, the fluorescent dye is selected from the group consistingof Alexa Flour 488, 532, 700, 750, Fluorescein, B-phycoerythrin,allophycocyanin, PBXL-3, and Qdot 605.

TABLE 2 FLUORESCENT ENTITIES Dye E Ex (nm) E (M)−1 Em (nm) MMw Bimane380 5,700 458 282.31 Dapoxyl 373 22,000 551 362.83 Dimethylaminocoumarin-4-acetic acid 375 22,000 470 344.32 Marina blue 365 19,000 460367.26 8-Anilino naphthalene-1-sulfonic acid 372 480 Cascade blue 37623,000 420 607.42 Alexa Fluor 405 402 35,000 421 1028.26 Cascade blue400 29,000 420 607.42 Cascade yellow 402 24,000 545 563.54 Pacific blue410 46,000 455 339.21 PyMPO 415 26,000 570 582.41 Alexa 430 433 15,000539 701.75 Atto-425 438 486 NBD 465 22,000 535 391.34 Alexa 488 49573,000 519 643.41 Fluorescein 494 79,000 518 376.32 Oregon Green 488 49676,000 524 509.38 Atto 495 495 522 Cy2 489 150,000 506 713.78 DY-480-XL500 40,000 630 514.60 DY-485-XL 485 20,000 560 502.59 DY-490-XL 48627,000 532 536.58 DY-500-XL 505 90,000 555 596.68 DY-520-XL 520 40,000664 514.60 Alexa Fluor 532 531 81,000 554 723.77 BODIPY 530/550 53477,000 554 513.31 6-HEX 535 98,000 556 680.07 6-JOE 522 75,000 550602.34 Rhodamine 6G 525 108,000 555 555.59 Atto-520 520 542 Cy3B 558130,000 572 658.00 Alexa Fluor 610 612 138,000 628 Alexa Fluor 633 632159,000 647 ca. 1200 Alexa Fluor 647 650 250,000 668 ca. 1250 BODIPY630/650 625 101,000 640 660.50 Cy5 649 250,000 670 791.99 Alexa Fluor660 663 110,000 690 Alexa Fluor 680 679 184,000 702 Alexa Fluor 700 702192,000 723 Alexa Fluor 750 749 240,000 782 B-phycoerythrin 546, 5652,410,000 575 240,000 R-phycoerythrin 480, 546, 1,960,000 578 240,000565 Allophycocyanin 650 700,000 660 700,000 PBXL-1 545 666 PBXL-3 614662 Atto-tec dyes Name Ex (nm) Em (nm) QY □ (ns) Atto 425 436 486 0.93.5 Atto 495 495 522 0.45 2.4 Atto 520 520 542 0.9 3.6 Atto 560 561 5850.92 3.4 Atto 590 598 634 0.8 3.7 Atto 610 605 630 0.7 3.3 Atto 655 665690 0.3 1.9 Atto 680 680 702 0.3 1.8 Dyomics Fluors Molar absorbance*molecular label Ex (nm) [l · mol−1 · cm−1] Em (nm) weight# [g · mol−1]DY-495/5 495 70,000 520 489.47 DY-495/6 495 70,000 520 489.47 DY-495X/5495 70,000 520 525.95 DY-495X/6 495 70,000 520 525.95 DY-505/5 50585,000 530 485.49 DY-505/6 505 85,000 530 485.49 DY-505X/5 505 85,000530 523.97 DY-505X/6 505 85,000 530 523.97 DY-550 553 122,000 578 667.76DY-555 555 100.000 580 636.18 DY-610 609 81.000 629 667.75 DY-615 621200.000 641 578.73 DY-630 636 200.000 657 634.84 DY-631 637 185.000 658736.88 DY-633 637 180.000 657 751.92 DY-635 647 175.000 671 658.86DY-636 645 190.000 671 760.91 DY-650 653 170.000 674 686.92 DY-651 653160.000 678 888.96 DYQ-660 660 117,000 — 668.86 DYQ-661 661 116,000 —770.90 DY-675 674 110.000 699 706.91 DY-676 674 145.000 699 807.95DY-680 690 125.000 709 634.84 DY-681 691 125.000 708 736.88 DY-700 70296.000 723 668.86 DY-701 706 115.000 731 770.90 DY-730 734 185.000 750660.88 DY-731 736 225.000 759 762.92 DY-750 747 240.000 776 712.96DY-751 751 220.000 779 814.99 DY-776 771 147.000 801 834.98 DY-780-OH770 70.000 810 757.34 DY-780-P 770 70.000 810 957.55 DY-781 783 98.000800 762.92 DY-782 782 102.000 800 660.88 EVOblue-10 651 101.440 664389.88 EVOblue-30 652 102.000 672 447.51 Quantum Dots: Qdot 525, 565,585, 605, 655, 705, 800

Suitable dyes for use in the invention include modified carbocyaninedyes. The modification of carbocyanine dyes includes the modification ofan indolium ring of the carbocyanine dye to permit a reactive group orconjugated substance at the number 3 position. The modification of theindolium ring provides dye conjugates that are uniformly andsubstantially more fluorescent on proteins, nucleic acids and otherbiopolymers, than conjugates labeled with structurally similarcarbocyanine dyes bound through the nitrogen atom at the number oneposition. In addition to having more intense fluorescence emission thanstructurally similar dyes at virtually identical wavelengths, anddecreased artifacts in their absorption spectra upon conjugation tobiopolymers, the modified carbocyanine dyes have greater photostabilityand higher absorbance (extinction coefficients) at the wavelengths ofpeak absorbance than the structurally similar dyes. Thus, the modifiedcarbocyanine dyes result in greater sensitivity in assays that use themodified dyes and their conjugates. Preferred modified dyes includecompounds that have at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. Other dye compoundsinclude compounds that incorporate an azabenzazolium ring moiety and atleast one sulfonate moiety. The modified carbocyanine dyes that can beused to detect individual molecules in various embodiments of theinvention are described in U.S. Pat. No. 6,977,305, which is hereinincorporated by reference in its entirety. Thus, in some embodiments thelabels of the invention utilize a fluorescent dye that includes asubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance group.

In some embodiments, the label comprises a fluorescent moiety thatincludes one or more Alexa dyes (Molecular Probes, Eugene, Oreg.). TheAlexa dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874;6,130,101; and 6,974,305 which are herein incorporated by reference intheir entirety. Some embodiments of the invention utilize a dye chosenfrom the group consisting of AlexaFluor 647, AlexaFluor 488, AlexaFluor532, AlexaFluor 555, AlexaFluor 610, AlexaFluor 680, AlexaFluor 700, andAlexaFluor 750. Some embodiments of the invention utilize a dye chosenfrom the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor647, AlexaFluor 700 and AlexaFluor 750. Some embodiments of theinvention utilize a dye chosen from the group consisting of AlexaFluor488, AlexaFluor 532, AlexaFluor 555, AlexaFluor 610, AlexaFluor 680,AlexaFluor 700, and AlexaFluor 750. Some embodiments of the inventionutilize the AlexaFluor 647 molecule, which has an absorption maximumbetween about 650 and 660 nm and an emission maximum between about 660and 670 nm. The AlexaFluor 647 dye is used alone or in combination withother AlexaFluor dyes.

In addition, currently available organic fluors can be improved byrendering them less hydrophobic by adding hydrophilic groups such aspolyethylene. Alternatively, currently sulfonated organic fluors such asthe AlexaFluor 647 dye can be rendered less acidic by making themzwitterionic. Particles such as antibodies that are labeled with themodified fluors are less likely to bind non-specifically to surfaces andproteins in immunoassays, and thus enable assays that have greatersensitivity and lower backgrounds. Methods for modifying and improvingthe properties of fluorescent dyes for the purpose of increasing thesensitivity of a system that detects single particles are known in theart. Preferably, the modification improves the Stokes shift whilemaintaining a high quantum yield.

2 Quantum Dots

In some embodiments, the fluorescent label moiety that is used to detecta molecule in a sample using the analyzer systems of the invention is aquantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which can bethought of the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One of the optical features of small excitonic QDs iscoloration, which is determined by the size of the dot. The larger thedot, the redder, or more towards the red end of the spectrum thefluorescence. The smaller the dot, the bluer or more towards the blueend it is. The bandgap energy that determines the energy and hence thecolor of the fluoresced light is inversely proportional to the square ofthe size of the QD. Larger QDs have more energy levels which are moreclosely spaced, thus allowing the QD to absorb photons containing lessenergy, i.e. those closer to the red end of the spectrum. Because theemission frequency of a dots dependent on the bandgap, it is thereforepossible to control the output wavelength of a dot with extremeprecision. In some embodiments the protein that is detected with thesingle particle analyzer system is labeled with a QD. In someembodiments, the single particle analyzer is used to detect a proteinlabeled with one QD and using a filter to allow for the detection ofdifferent proteins at different wavelengths.

QDs have broad excitation and narrow emission properties which when usedwith color filtering require only a single electromagnetic source formultiplex analysis of multiple targets in a single sample to resolveindividual signals. Thus, in some embodiments, the analyzer systemcomprises one continuous wave laser and particles that are each labeledwith one QD. Colloidally prepared QDs are free floating and can beattached to a variety of molecules via metal coordinating functionalgroups. These groups include but are not limited to thiol, amine,nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acidsor other ligands. By bonding appropriate molecules to the surface, thequantum dots can be dispersed or dissolved in nearly any solvent orincorporated into a variety of inorganic and organic films. Quantum dots(QDs) can be coupled to streptavidin directly through a male imide estercoupling reaction or to antibodies through a meleimide-thiol couplingreaction. This yields a material with a biomolecule covalently attachedon the surface, which produces conjugates with high specific activity.In some embodiments, the protein that is detected with the singleparticle analyzer is labeled with one quantum dot. In some embodimentsthe quantum dot is between 10 and 20 nm in diameter. In otherembodiments, the quantum dot is between 2 and 10 nm in diameter. UsefulQuantum Dots include QD 605, QD 610, QD 655, and QD 705. A particularlypreferred Quantum Dot is QD 605.

C. Binding Partner-Fluorescent Moiety Compositions

The labels of the invention generally contain a binding partner, e.g.,antibody, bound to a fluorescent moiety to provide the requisitefluorescence for detection and quantitation in the instruments describedherein. Any suitable combination of binding partner and fluorescentmoiety for detection in the single molecule detectors described hereinmay be used as a label in the invention. In some embodiments, theinvention provides a label for a marker of a biological state, where thelabel includes an antibody to the marker and a fluorescent moiety. Themarker may be any of the markers described above. The antibody may beany antibody as described above. A fluorescent moiety may be attachedsuch that the label is capable of emitting an average of at least about50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600,700, 800, 900, or 1000, photons when simulated by a laser emitting lightat the excitation wavelength of the moiety, where the laser is focusedon a spot of not less than about 5 microns in diameter that contains thelabel, and wherein the total energy directed at the spot by the laser isno more than about 3 microJoules. In some embodiments, the fluorescentmoiety may be a fluorescent moiety that is capable of emitting anaverage of at least about 50, 100, 150, or 200 photons when simulated bya laser emitting light at the excitation wavelength of the moiety, wherethe laser is focused on a spot of not less than about 5 microns indiameter that contains the moiety, and wherein the total energy directedat the spot by the laser is no more than about 3 microJoules. Thefluorescent moiety may be a fluorescent moiety that includes one or moredye molecules with a structure that includes a substituted indolium ringsystem in which the substituent on the 3-carbon of the indolium ringcontains a chemically reactive group or a conjugated substance group.The label composition may include a fluorescent moiety that includes oneor more dye molecules selected from the group consisting of AlexaFluor488, 532, 647, 700, or 750. The label composition may include afluorescent moiety that includes one or more dye molecules selected fromthe group consisting of AlexaFluor 488, 532, 700, or 750. The labelcomposition may include a fluorescent moiety that includes one or moredye molecules that are AlexaFluor 488. The label composition may includea fluorescent moiety that includes one or more dye molecules that areAlexaFluor 555. The label composition may include a fluorescent moietythat includes one or more dye molecules that are AlexaFluor 610. Thelabel composition may include a fluorescent moiety that includes one ormore dye molecules that are AlexaFluor 647. The label composition mayinclude a fluorescent moiety that includes one or more dye moleculesthat are AlexaFluor 680. The label composition may include a fluorescentmoiety that includes one or more dye molecules that are AlexaFluor 700.The label composition may include a fluorescent moiety that includes oneor more dye molecules that are AlexaFluor 750.

In some embodiments the invention provides a composition for thedetection of a marker of a biological state that includes an AlexFluormolecule, e.g. an AlexaFluor molecule selected from the describedgroups, such as an AlexaFluor 647 molecule attached to a to an antibodyspecific for the marker. In some embodiments the composition includes anaverage of 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 to 6,or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, orabout 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, orabout 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 AlexaFluor647 molecules molecule attached to an antibody for the marker. In someembodiments the invention provides a composition for the detection amarker of a biological state that includes an average of 1 to 11, orabout 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, orabout 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, orabout 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, orabout 2, 3, 4, 5, 6, or more than about 6 AlexaFluor 647 moleculesmolecule attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 2 to 10AlexaFluor 647 molecules molecule attached to an antibody specific tothe marker. In some embodiments the invention provides a composition forthe detection of a marker of a biological state that includes an averageof about 2 to 8 AlexaFluor 647 molecules molecule attached to anantibody specific to the marker. In some embodiments the inventionprovides a composition for the detection of a marker of a biologicalstate that includes an average of about 2 to 6 AlexaFluor 647 moleculesmolecule attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 2 to 4AlexaFluor 647 molecules molecule attached to an antibody specific tothe marker. In some embodiments the invention provides a composition forthe detection of a marker of a biological state that includes an averageof about 3 to 8 AlexaFluor 647 molecules molecule attached to anantibody specific to the marker. In some embodiments the inventionprovides a composition for the detection of a marker of a biologicalstate that includes an average of about 3 to 6 AlexaFluor 647 moleculesmolecule attached to an antibody specific to the marker. In someembodiments the invention provides a composition for the detection of amarker of a biological state that includes an average of about 4 to 8AlexaFluor 647 molecules molecule attached to an antibody specific tothe marker.

Attachment of the fluorescent moiety, or fluorescent entities that makeup the fluorescent moiety, to the binding partner, e.g., antibody, maybe by any suitable means; such methods are well-known in the art andexemplary methods are given in the Examples. In some embodiments, afterattachment of the fluorescent moiety to the binding partner to form alabel for use in the methods of the invention, and prior to the use ofthe label for labeling the protein of interest, it is useful to performa filtration step. E.g., an antibody-dye label may be filtered prior touse, e.g., through a 0.2 micron filter, or any suitable filter forremoving aggregates. Other reagents for use in the assays of theinvention may also be filtered, e.g., e.g., through a 0.2 micron filter,or any suitable filter. Without being bound by theory, it is thoughtthat such filtration removes a portion of the aggregates of the, e.g.,antibody-dye labels. As such aggregates will bind as a unit to theprotein of interest, but upon release in elution buffer are likely todisaggregate, false positives may result; i.e., several labels will bedetected from an aggregate that has bound to only a single proteinmolecule of interest. Regardless of theory, filtration has been found toreduce false positives in the subsequent assay and to improve accuracyand precision.

It will be appreciated that immunoassays often employ a sandwich format,in which binding partner pairs, e.g. antibodies, to the same molecule,e.g., marker are used. The invention also encompasses binding partnerpairs, e.g., antibodies, wherein both antibodies are specific to thesame molecule, e.g., the same marker, and wherein at least one member ofthe pair is a label as described herein. Thus, for any label thatincludes a binding-partner and a fluorescent moiety, the invention alsoencompasses a pair of binding partners wherein the first bindingpartner, e.g., antibody, is part of the label, and the second bindingpartner, e.g., antibody, is, typically, unlabeled and serves as acapture binding partner. In addition, binding partner pairs arefrequently used in FRET assays. FRET assays useful in the invention aredisclosed in U.S. patent application Ser. No. 11/048,660, incorporatedby reference herein in its entirety, and the present invention alsoencompasses binding partner pairs, each of which includes a FRET label.

IV. Highly Sensitive Analysis of Molecules

In one aspect, the invention provides a method for determining thepresence or absence of a single molecule, e.g., a molecule of a markerof a biological state, in a sample, by i) labeling the molecule ifpresent, with a label; and ii) detecting the presence or absence of thelabel, where the detection of the presence of the label indicates thepresence of the single molecule in the sample. In some embodiments, themethod is capable of detecting the molecule at a limit of detection ofless than about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5,4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2. 0.1, 0.05, 0.01,0.005, or 0.001 femtomolar. In some embodiments, the method is capableof detecting the molecule at a limit of detection of less than about 100femtomolar. In some embodiments, the method is capable of detecting themolecule at a limit of detection of less than about 10 femtomolar. Insome embodiments, the method is capable of detecting the molecule at alimit of detection of less than about 1 femtomolar. In some embodiments,the method is capable of detecting the molecule at a limit of detectionof less than about 0.1 femtomolar. In some embodiments, the method iscapable of detecting the molecule at a limit of detection of less thanabout 0.01 femtomolar. In some embodiments, the method is capable ofdetecting the molecule at a limit of detection of less than about 0.001femtomolar. Detection limits may be determined by use of an appropriatestandard, e.g., National Institute of Standards and Technology referencestandard material.

The methods also provide methods of determining a concentration of amolecule, e.g., a marker of a biological state, in a sample by detectingsingle molecules of the molecule in the sample. The “detecting” of asingle molecule includes detecting the molecule directly or indirectly.In the case of indirect detection, labels that corresponds to singlemolecules, e.g., a labels that have been attached to the singlemolecules, may be detected.

In some embodiments, the invention provides a method for determining thepresence or absence of a single molecule of a protein in a biologicalsample, comprising labeling said molecule with a label and detecting thepresence or absence of said label in a single molecule detector, whereinsaid label comprises a fluorescent moiety that is capable of emitting atleast about 200 photons when simulated by a laser emitting light at theexcitation wavelength of the moiety, wherein the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and wherein the total energy directed at the spot by the laser is nomore than about 3 microJoules. The single molecule detector may, in someembodiments, comprise not more than one interrogation space. The limitof detection of the single molecule in the sample may be less than about10, 1, 0.1, 0.01, or 0.001 femtomolar. In some embodiments, the limit ofdetection is less than about 1 femtomolar. The detecting may comprisedetecting electromagnetic radiation emitted by said fluorescent moiety.The method may further comprise exposing said fluorescent moiety toelectromagnetic radiation, e.g., electromagnetic radiation provided by alaser, such as a laser with a power output of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mW. In some embodiments,the laser stimulates provides light to the interrogation space for aduration of about 10-1000 microseconds, or about 1000, 250, 100, 50, 25or 10 microseconds. In some embodiments, the label further comprises abinding partner specific for binding said molecule, such as an antibody.In some embodiments, the fluorescent moiety comprises a fluorescent dyemolecule, such as a dye molecule that comprises at least one substitutedindolium ring system in which the substituent on the 3-carbon of theindolium ring contains a chemically reactive group or a conjugatedsubstance. In some embodiments, the dye molecule is an AlexFluormolecule selected from the group consisting of AlexaFluor 488,AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. Insome embodiments, the dye molecule is an AlexaFluor647 dye molecule. Insome embodiments, the fluorescent moiety comprises a plurality ofAlexaFluor 647 molecules. In some embodiments, the plurality ofAlexaFluor 647 molecules comprises about 2-4 AlexaFluor 647 molecules,or about 3-6 AlexaFluor 647 molecules. In some embodiments, thefluorescent moiety is a quantum dot. The method may further comprisemeasuring the concentration of said protein in the sample.

In some embodiments, detecting the presence or absence of said labelcomprises: (i) passing a portion of said sample through an interrogationspace; (ii) subjecting said interrogation space to exposure toelectromagnetic radiation, said electromagnetic radiation beingsufficient to stimulate said fluorescent moiety to emit photons, if saidlabel is present; and (iii) detecting photons emitted during saidexposure of step (ii). The method may further comprise determining abackground photon level in said interrogation space, wherein saidbackground level represents the average photon emission of theinterrogation space when it is subjected to electromagnetic radiation inthe same manner as in step (ii), but without label in the interrogationspace. The method may further comprise comparing the amount of photonsdetected in step (iii) to a threshold photon level, wherein saidthreshold photon level is a function of said background photon level,wherein an amount of photons detected in step (iii) greater that thethreshold level indicates the presence of said label, and an amount ofphotons detected in step (iii) equal to or less than the threshold levelindicates the absence of said label.

A. Sample

The sample may be any suitable sample. Typically, the sample is abiological sample, e.g., a biological fluid. Such fluids include,without limitation, bronchioalveolar lavage fluid (BAL), blood, serum,plasma, urine, nasal swab, cerebrospinal fluid, pleural fluid, synovialfluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph fluid,interstitial fluid, tissue homogenate, cell extracts, saliva, sputum,stool, physiological secretions, tears, mucus, sweat, milk, semen,seminal fluid, vaginal secretions, fluid from ulcers and other surfaceeruptions, blisters, and abscesses, and extracts of tissues includingbiopsies of normal, malignant, and suspect tissues or any otherconstituents of the body which may contain the target particle ofinterest. Other similar specimens such as cell or tissue culture orculture broth are also of interest. In some embodiments, the sample is ablood sample. In some embodiments the sample is a plasma sample. In someembodiments the sample is a serum sample. In some embodiments, thesample is a urine sample. In some embodiments, the sample is a nasalswab.

B. Sample Preparation

In general, any method of sample preparation may be used that produces alabel corresponding to a molecule of interest, e.g., a marker of abiological state, that is wished to be measured, where the label isdetectable in the instruments described herein. As is known in the art,sample preparation in which a label is added to one or more moleculesmay be performed in a homogeneous or heterogeneous format. In someembodiments, the sample preparation is formed in a homogenous format. Inanalyzer system employing a homogenous format, unbound label is notremoved from the sample. See, e.g., U.S. patent application Ser. No.11/048,660. In some embodiments, the particle or particles of interestare labeled by addition of labeled antibody or antibodies that bind tothe particle or particles of interest.

In some embodiments, a heterogeneous assay format is used, where,typically, a step is employed for removing unbound label. Such assayformats are well-known in the art. One particularly useful assay formatis a sandwich assay, e.g., a sandwich immunoassay. In this format, themolecule of interest, e.g., marker of a biological state, is captured,e.g., on a solid support, using a capture binding partner. Unwantedmolecules and other substances may then optionally be washed away,followed by binding of a label comprising a detection binding partnerand a detectable label, e.g., fluorescent moiety. Further washes removeunbound label, then the detectable label is released, usually though notnecessarily still attached to the detection binding partner. Inalternative embodiments, sample and label are added to the capturebinding partner without a wash in between, e.g., at the same time. Othervariations will be apparent to one of skill in the art.

In some embodiments, the method for detecting the molecule of interest,e.g., marker of a biological state, uses a sandwich assay withantibodies, e.g., monoclonal antibodies as capture binding partners. Themethod comprises binding molecules in a sample to a capture antibodythat is immobilized on a binding surface, and binding the labelcomprising a detection antibody to the molecule to form a “sandwich”complex. The label comprises the detection antibody and a fluorescentmoiety, as described herein, which is detected, e.g., using the singlemolecule analyzers of the invention. Both the capture and detectionantibodies specifically bind the molecule. Many example of sandwichimmunoassays are known, and some are described in U.S. Pat. No.4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al.,both of which are incorporated herein by reference. Further examplesspecific to specific markers are described in the Examples.

The capture binding partner may be attached to a solid support, e.g., amicrotiter plate or paramagnetic beads. In some embodiments, theinvention provides a binding partner for a molecule of interest, e.g.,marker of a biological state, attached to a paramagnetic bead. Anysuitable binding partner that is specific for the molecule that it iswished to capture may be used. The binding partner may be an antibody,e.g., a monoclonal antibody. Production and sources of antibodies aredescribed elsewhere herein. It will be appreciated that antibodiesidentified herein as useful as a capture antibody may also be useful asdetection antibodies, and vice versa.

The attachment of the binding partner, e.g., antibody, to the solidsupport may be covalent or noncovalent. In some embodiments, theattachment is noncovalent. An example of a noncovalent attachmentwell-known in the art is biotin-avidin/streptavidin interactions. Thus,in some embodiments, a solid support, e.g., a microtiter plate or aparamagnetic bead, is attached to the capture binding partner, e.g.,antibody, through noncovalent attachment, e.g.,biotin-avidin/streptavidin interactions. In some embodiments, theattachment is covalent. Thus, in some embodiments, a solid support,e.g., a microtiter plate or a paramagnetic bead, is attached to thecapture binding partner, e.g., antibody, through covalent attachment.

Covalent attachment in which the orientation of the capture antibody issuch that capture of the molecule of interest is optimized is especiallyuseful. For example, in some embodiments a solid support, e.g., amicrotiter plate or a paramagnetic microparticle, may be used in whichthe attachment of the binding partner, e.g., antibody, is an orientedattachment, e.g., a covalent oriented attachment.

An exemplary protocol for oriented attachment of an antibody to a solidsupport is as follows: IgG is dissolved in 0.1M sodium acetate buffer,pH 5.5 to a final concentration of 1 mg/ml. An equal volume of ice-cold20 mM sodium periodate in 0.1 M sodium acetate, pH 5.5 is added. The IgGis allowed to oxidize for ½ hour on ice. Excess periodate reagent isquenched by the addition of 0.15 volume of 1 M glycerol. Low molecularweight byproducts of the oxidation reaction are removed byultrafiltration. The oxidized IgG fraction is diluted to a suitableconcentration (typically 0.5 micrograms IgG per ml) and reacted withhydrazide-activated multiwell plates for at least two hours at roomtemperature. Unbound IgG is removed by washing the multiwell plate withborate buffered saline or another suitable buffer. The plate may bedried for storage, if desired. A similar protocol may be followed formicrobeads if the material of the microbead is suitable for suchattachment.

In some embodiments, the solid support is a microliter plate. In someembodiments, the solid support is a paramagnetic bead. An exemplaryparamagnetic bead is Streptavidin C1 (Dynal, 650.01-03). Other suitablebeads will be apparent to those of skill in the art. Methods forattachment of antibodies to paramagnetic beads are well-known in theart. One example is given in Example 2.

The molecule of interest is contacted with the capture binding partner,e.g., capture antibody immobilized on a solid support. Some samplepreparation may be used; e.g., preparation of serum from blood samplesor concentration procedures before the sample is contacted with thecapture antibody. Protocols for binding of proteins in immunoassays arewell-known in the art and are included in the Examples.

The time allowed for binding will vary depending on the conditions; itwill be apparent that shorter binding times are desirable in somesettings, especially in a clinical setting. The use of, e.g.,paramagnetic beads can reduce the time required for binding. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., antibody, is less that about 12, 10,8, 6, 4, 3, 2, or 1 hours, or less than about 60, 50, 40, 30, 25, 20,15, 10, or 5 minutes. In some embodiments, the time allowed for bindingof the molecule of interest to the capture binding partner, e.g.,antibody, is less than about 60 minutes. In some embodiments, the timeallowed for binding of the molecule of interest to the capture bindingpartner, e.g., antibody, is less that about 40 minutes. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., antibody, is less that about 30minutes. In some embodiments, the time allowed for binding of themolecule of interest to the capture binding partner, e.g., antibody, isless that about 20 minutes. In some embodiments, the time allowed forbinding of the molecule of interest to the capture binding partner,e.g., antibody, is less that about 15 minutes. In some embodiments, thetime allowed for binding of the molecule of interest to the capturebinding partner, e.g., antibody, is less that about 10 minutes. In someembodiments, the time allowed for binding of the molecule of interest tothe capture binding partner, e.g., antibody, is less that about 5minutes.

In some embodiments, following the binding of the troponin particles tothe capture binding partner, e.g., capture antibody, particles that mayhave bound nonspecifically, as well as other unwanted substances in thesample, are washed away leaving substantially only specifically boundtroponin particles. In other embodiments, no wash is used betweenadditions of sample and label; it will be appreciated that this reducessample preparation time even further. Thus, in some embodiments, thetime allowed for both binding of the molecule of interest to the capturebinding partner, e.g., antibody, and binding of the label to themolecule of interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less that about 60 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 40 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 30 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 20 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 15 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 10 minutes.In some embodiments, the time allowed for both binding of the moleculeof interest to the capture binding partner, e.g., antibody, and bindingof the label to the molecule of interest, is less than about 5 minutes.

Some immunoassay diagnostic reagents including the capture and signalantibodies used to measure the molecule of interest may be derived fromthe sera of animals. Endogenous human heterophilic antibodies, or humananti-animal antibodies, which have the ability to bind toimmunoglobulins of other species, are present in the serum or plasma ofmore than 10% of patients. These circulating heterophile antibodies mayinterfere with immunoassay measurements. In sandwich immunoassays, theseheterophilic antibodies can either bridge the capture and detection(diagnostic) antibodies, thereby producing a false-positive signal, orthey may block the binding of the diagnostic antibodies, therebyproducing a false-negative signal. In competitive immunoassays, theheterophile antibodies may bind to the analytic antibody and inhibit itsbinding to the troponin. They also may either block or augment theseparation of the antibody-troponin complex from free troponin,especially when antispecies antibodies are used in the separationsystems. Therefore, the impact of these heterophile antibodyinterferences are difficult to predict. Thus, it would be advantageousto block the binding of any heterophilic antibodies. In some embodimentsof the invention, the immunoassay includes the step of depleting thesample of heterophile antibodies using one or more heterophile antibodyblockers. Methods for removing heterophile antibodies from samples thatare to be tested in immunoassays are known and include: heating thespecimen in a sodium acetate buffer, pH 5.0, for 15 minutes at 90° C.and centrifuging at 1200 g for 10 minutes, or the heterophile antibodiescan be precipitated using polyethylene glycol (PEG); immunoextractingthe interfering heterophile immunoglobulins from the specimen usingprotein A or protein G; or adding nonimmune mouse IgG. Embodiments ofthe methods of the invention contemplate preparing the sample prior toanalysis with the single molecule detector. The appropriateness of themethod of pretreatment may be determined. Biochemicals to minimizeimmunoassay interference caused by heterophile antibodies arecommercially available. For example, a product called MAK33, which is anIgG1 monoclonal antibody to h-CK-MM, may be obtained from BoehringerMannheim. The MAK33 plus product contains a combination of IgG1 andIgG1-Fab. The polyMAK33 contains IgG1-Fab polymerized with IgG1, and thepolyMAC 2b/2a contains IgG2a-Fab polymerized with IgG2b. A secondcommercial source of biochemicals to neutralize heterophile antibodiesis Immunoglobulin Inhibiting Reagent marketed by Bioreclamation Inc,East Meadow, N.Y. This product is a preparation of immunoglobulins (IgGand IgM) from multiple species, mainly murine IgG2a, IgG2b, and IgG3from Balb/c mice. In some embodiments the heterophile antibody may beimmunoextracted from the sample using methods known in the art e.g.depleting the sample of the heterophile antibody by binding theinterfering antibody to protein A or G. In some embodiments, theheterophile antibody is neutralized using one or more heterophileantibody blockers. Heterophile blockers may be selected from the groupconsisting of anti-isotype heterophile antibody blockers, anti-idiotypeheterophile antibody blockers, and anti-anti-idiotype heterophileantibody blockers. In some embodiments a combination of heterophileantibody blockers may be used.

Label is added either with or following the addition of sample andwashing. Protocols for binding of antibody and other immunolabels toproteins and other molecules are well-known in the art. If the labelbinding step is separate from capture binding, the time allowed forlabel binding can be important, e.g., in the clinical setting. In someembodiments, the time allowed for binding of the molecule of interest tothe label, e.g., antibody-dye, is less than about 12, 10, 8, 6, 4, 3, 2,or 1 hours, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5minutes. In some embodiments, the time allowed for binding of themolecule of interest to the label, e.g., antibody-dye, is less thanabout 60 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 40 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 30 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 20 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 15 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 10 minutes. In some embodiments, the time allowed for binding ofthe molecule of interest to the label, e.g., antibody-dye, is less thanabout 5 minutes. Excess label is removed by washing.

Label is then eluted from the protein of interest. Preferred elutionbuffers are effective in releasing the label without generatingsignificant background. It is also useful if the elution buffer isbacteriostatic. Elution buffers of use in the invention include achaotrope, e.g., urea or a guanidinium compound; a buffer, e.g., boratebuffered saline; a protein carrier, e.g., an albumin, such as human,bovine, or fish albumin, or an IgG, to coat the wall of the capillarytube in the detection instrument; and a surfactant, e.g., an ionic ornonionic detergent, selected so as to produce a relatively lowbackground, e.g., Tween 20, Triton X-100, or SDS.

The elution buffer/label aliquot that is sampled into the singlemolecule detector is referred to as the “processing sample,” todistinguish it from the original sample which was obtained from anindividual.

In another embodiment, the solid phase binding assay may employ acompetitive binding assay format. One such method comprises a)competitively binding to a capture antibody immobilized on a bindingsurface i) a molecule of interest, e.g., marker of a biological state,in a sample and ii) a labeled analog of the molecule comprising adetectable label (the detection reagent) and b) measuring the amount ofthe label using a single particle analyzer. Another such methodcomprises a) competitively binding to an antibody having a detectablelabel (the detection reagent) i) a molecule of interest, e.g., marker ofa biological state in a sample and ii) an analog of the molecule that isimmobilized on a binding surface (the capture reagent) and b) measuringthe amount of the label using a single particle analyzer. An “analog ofa molecule” refers, herein, to a species that competes with a moleculefor binding to a capture antibody. Examples of competitive immunoassaysare disclosed in U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat.No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al.,all of which are incorporated herein by reference.

C. Detection of Molecule of Interest and Determination of Concentration

Following elution, the label is run through a single molecule detectorin e.g., the elution buffer. A processing sample may contain no label, asingle label, or a plurality of labels. The number of labels correspondsor is proportional to (if dilutions or fractions of samples are used)the number of molecules of the molecule of interest, e.g., marker of abiological state captured during the capture step.

Any suitable single molecule detector capable of detecting the labelused with the molecule of interest may be used. Suitable single moleculedetectors are described herein. Typically the detector will be part of asystem that includes an automatic sampler for sampling prepared samples,and, optionally, a recovery system to recover samples.

In some embodiments, the processing sample is analyzed in a singlemolecule analyzer that utilizes a capillary flow system, and thatincludes a capillary flow cell, a laser to illuminate an interrogationspace in the capillary through which processing sample is passed, adetector to detect radiation emitted from the interrogation space, and asource of motive force to move a processing sample through theinterrogation space. In some embodiments, the single molecule analyzerfurther comprises a microscope objective lens that collects lightemitted from processing sample as it passes through the interrogationspace, e.g., a high numerical aperture microscope objective. In someembodiments, the laser and detector are in a confocal arrangement. Insome embodiments, the laser is a continuous wave laser. In someembodiments, the detector is a avalanche photodiode detector. In someembodiments, the source of motive force is a pump to provide pressure.In some embodiments, the invention provides an analyzer system thatincludes a sampling system capable of automatically sampling a pluralityof samples providing a fluid communication between a sample containerand the interrogation space. In some embodiments, the interrogationspace has a volume of between about 0.001 and 500 pL, or between about0.01 pL and 100 pL, or between about 0.01 pL and 10 pL, or between about0.01 pL and 5 pL, or between about 0.01 pL and 0.5 pL, or between about0.02 pL and about 300 pL, or between about 0.02 pL and about 50 pL orbetween about 0.02 pL and about 5 pL or between about 0.02 pL and about0.5 pL or between about 0.02 pL and about 2 pL, or between about 0.05 pLand about 50 pL, or between about 0.05 pL and about 5 pL, or betweenabout 0.05 pL and about 0.5 pL, or between about 0.05 pL and about 0.2pL, or between about 0.1 pL and about 25 pL. In some embodiments, theinterrogation space has a volume between about 0.004 pL and 100 pL. Insome embodiments, the interrogation space has a volume between about0.02 pL and 50 pL. In some embodiments, the interrogation space has avolume between about 0.001 pL and 10 pL. In some embodiments, theinterrogation space has a volume between about 0.001 pL and 10 pL. Insome embodiments, the interrogation space has a volume between about0.01 pL and 5 pL. In some embodiments, the interrogation space has avolume between about 0.02 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.05 pL and 5 pL. In someembodiments, the interrogation space has a volume between about 0.05 pLand 10 pL. In some embodiments, the interrogation space has a volumebetween about 0.5 pL and about 5 pL. In some embodiments, theinterrogation space has a volume between about 0.02 pL and about 0.5 pL.

In some embodiments, the single molecule detector used in the methods ofthe invention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation space, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.02 pL and about 50 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.004 pL and about100 pL. In some embodiments, the single molecule detector used in themethods of the invention utilizes a capillary flow system, and includesa capillary flow cell, a continuous wave laser to illuminate aninterrogation space in the capillary through which processing sample ispassed, a high numerical aperture microscope objective lens thatcollects light emitted from processing sample as it passes through theinterrogation space wherein the lens has a numerical aperture of atleast about 0.8, an avalanche photodiode detector to detect radiationemitted from the interrogation space, and a pump to provide pressure tomove a processing sample through the interrogation space, where theinterrogation space is between about 0.05 pL and about 10 pL. In someembodiments, the single molecule detector used in the methods of theinvention utilizes a capillary flow system, and includes a capillaryflow cell, a continuous wave laser to illuminate an interrogation spacein the capillary through which processing sample is passed, a highnumerical aperture microscope objective lens that collects light emittedfrom processing sample as it passes through the interrogation spacewherein the lens has a numerical aperture of at least about 0.8, anavalanche photodiode detector to detect radiation emitted from theinterrogation space, and a pump to provide pressure to move a processingsample through the interrogation space, where the interrogation space isbetween about 0.05 pL and about 5 pL. In some embodiments, the singlemolecule detector used in the methods of the invention utilizes acapillary flow system, and includes a capillary flow cell, a continuouswave laser to illuminate an interrogation space in the capillary throughwhich processing sample is passed, a high numerical aperture microscopeobjective lens that collects light emitted from processing sample as itpasses through the interrogation space wherein the lens has a numericalaperture of at least about 0.8, an avalanche photodiode detector todetect radiation emitted from the interrogation space, and a pump toprovide pressure to move a processing sample through the interrogationspace, where the interrogation space is between about 0.5 pL and about 5pL. In any of these embodiments the analyzer may contain not more thanone interrogation space.

In some embodiments, the single molecule detector is capable ofdetermining a concentration for a molecule of interest in a sample wheresample may range in concentration over a range of at least about100-fold, or 1000-fold, or 10,000-fold, or 100,000-fold, or 300,00-fold,or 1,000,000-fold, or 10,000,000-fold, or 30,000,000-fold.

In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 50%,40%, 30%, 20%, 15%, or 10% in concentration of an analyte between afirst sample and a second sample that are introduced into the detector,where the volume of the first sample and said second sample introducedinto the analyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30,20, 15, 10, 5, 4, 3, 2, or 1 ul, and wherein the analyte is present at aconcentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20,15, 10, 5, 4, 3, 2, or 1 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 50% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 100 ul, and wherein theanalyte is present at a concentration of less than about 100 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 40%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 50 ul, and wherein the analyte is present at a concentrationof less than about 50 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 20 ul, and wherein theanalyte is present at a concentration of less than about 20 femtomolar.In some embodiments, the methods of the invention utilize a singlemolecule detector capable detecting a difference of less than about 20%in concentration of an analyte between a first sample and a secondsample that are introduced into the detector, where the volume of thefirst sample and said second sample introduced into the analyzer is lessthan about 10 ul, and wherein the analyte is present at a concentrationof less than about 10 femtomolar. In some embodiments, the methods ofthe invention utilize a single molecule detector capable detecting adifference of less than about 20% in concentration of an analyte betweena first sample and a second sample that are introduced into thedetector, where the volume of the first sample and said second sampleintroduced into the analyzer is less than about 5 ul, and wherein theanalyte is present at a concentration of less than about 5 femtomolar.

The single molecule detector and systems are described in more detailbelow. Further embodiments of single molecule analyzers useful in themethods of the invention, such as detectors with more than oneinterrogation window, detectors utilize electrokinetic orelectrophoretic flow, and the like, may be found in U.S. patentapplication Ser. No. 11/048,660, incorporated by reference herein in itsentirety.

Between runs the instrument may be washed. A wash buffer that maintainsthe salt and surfactant concentrations of the sample may be used in someembodiments to maintain the conditioning of the capillary; i.e., to keepthe capillary surface relatively constant between samples to reducevariability.

A feature that contributes to the extremely high sensitivity of theinstruments and methods of the invention is the method of detecting andcounting labels, which, in some embodiments, are attached to singlemolecules to be detected or, more typically, correspond to a singlemolecule to be detected. Briefly, the processing sample flowing throughthe capillary is effectively divided into a series of detection events,by subjecting a given interrogation space of the capillary to EMradiation from a laser that emits light at an appropriate excitationwavelength for the fluorescent moiety used in the label for apredetermined period of time, and detecting photons emitted during thattime. Each predetermined period of time is a “bin.” If the total numberof photons detected in a given bin exceeds a predetermined thresholdlevel, a detection event is registered for that bin, i.e., a label hasbeen detected. If the total number of photons is not at thepredetermined threshold level, no detection event is registered. In someembodiments, processing sample concentration is dilute enough that, fora large percentage of detection events, the detection event representsonly one label passing through the window, which corresponds to a singlemolecule of interest in the original sample, that is, few detectionevents represent more than one label in a single bin. In someembodiments, further refinements are applied to allow greaterconcentrations of label in the processing sample to be detectedaccurately, i.e., concentrations at which the probability of two or morelabels being detected as a single detection event is no longerinsignificant.

Although other bin times may be used without departing from the scope ofthe present invention, in some embodiments the bin times are selected inthe range of about 1 microsecond to about 5 ms. In some embodiments, thebin time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750,800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In someembodiments, the bin time is less than about 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600,700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. Insome embodiments, the bin time is about 1 to 1000 microseconds. In someembodiments, the bin time is about 1 to 750 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 250 microseconds. In someembodiments, the bin time is about 1 to 100 microseconds. In someembodiments, the bin time is about 1 to 50 microseconds. In someembodiments, the bin time is about 1 to 40 microseconds. In someembodiments, the bin time is about 1 to 30 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 20 microseconds. In someembodiments, the bin time is about 1 to 10 microseconds. In someembodiments, the bin time is about 1 to 500 microseconds. In someembodiments, the bin time is about 1 to 5 microseconds. In someembodiments, the bin time is about 5 to 500 microseconds. In someembodiments, the bin time is about 5 to 250 microseconds. In someembodiments, the bin time is about 5 to 100 microseconds. In someembodiments, the bin time is about 5 to 50 microseconds. In someembodiments, the bin time is about 5 to 20 microseconds. In someembodiments, the bin time is about 5 to 10 microseconds. In someembodiments, the bin time is about 10 to 500 microseconds. In someembodiments, the bin time is about 10 to 250 microseconds. In someembodiments, the bin time is about 10 to 100 microseconds. In someembodiments, the bin time is about 10 to 50 microseconds. In someembodiments, the bin time is about 10 to 30 microseconds. In someembodiments, the bin time is about 10 to 20 microseconds. In someembodiments, the bin time is about 5 microseconds. In some embodiments,the bin time is about 5 microseconds. In some embodiments, the bin timeis about 6 microseconds. In some embodiments, the bin time is about 7microseconds. In some embodiments, the bin time is about 8 microseconds.In some embodiments, the bin time is about 9 microseconds. In someembodiments, the bin time is about 10 microseconds. In some embodiments,the bin time is about 11 microseconds. In some embodiments, the bin timeis about 12 microseconds. In some embodiments, the bin time is about 13microseconds. In some embodiments, the bin time is about 14microseconds. In some embodiments, the bin time is about 5 microseconds.In some embodiments, the bin time is about 15 microseconds. In someembodiments, the bin time is about 16 microseconds. In some embodiments,the bin time is about 17 microseconds. In some embodiments, the bin timeis about 18 microseconds. In some embodiments, the bin time is about 19microseconds. In some embodiments, the bin time is about 20microseconds. In some embodiments, the bin time is about 25microseconds. In some embodiments, the bin time is about 30microseconds. In some embodiments, the bin time is about 40microseconds. In some embodiments, the bin time is about 50microseconds. In some embodiments, the bin time is about 100microseconds. In some embodiments, the bin time is about 250microseconds. In some embodiments, the bin time is about 500microseconds. In some embodiments, the bin time is about 750microseconds. In some embodiments, the bin time is about 1000microseconds.

In some embodiments, the background noise level is determined from themean noise level, or the root-mean-square noise. In other cases, atypical noise value or a statistical value is chosen. In most cases, thenoise is expected to follow a Poisson distribution. Thus, in someembodiments, determining the concentration of a particle-label complexin a sample comprises determining the background noise level.

Thus, as a label flows through the capillary flow cell, it is irradiatedby the laser beam to generate a burst of photons. The photons emitted bythe label are discriminated from background light or background noiseemission by considering only the bursts of photons that have energyabove a predetermined threshold energy level which accounts for theamount of background noise that is present in the sample. Backgroundnoise typically comprises low frequency emission produced, for example,by the intrinsic fluorescence of non-labeled particles that are presentin the sample, the buffer or diluent used in preparing the sample foranalysis, Raman scattering and electronic noise. In some embodiments,the value assigned to the background noise is calculated as the averagebackground signal noise detected in a plurality of bins, which aremeasurements of photon signals that are detected in an interrogationspace during a predetermined length of time. Thus in some embodiments,background noise is calculated for each sample as a number specific tothat sample.

Given the value for the background noise, the threshold energy level canbe assigned. As discussed above, the threshold value is determined todiscriminate true signals (due to fluorescence of a label) from thebackground noise. Care must be taken in choosing a threshold value suchthat the number of false positive signals from random noise is minimizedwhile the number of true signals which are rejected is also minimized.Methods for choosing a threshold value include determining a fixed valueabove the noise level and calculating a threshold value based on thedistribution of the noise signal. In one embodiment, the threshold isset at a fixed number of standard deviations above the background level.Assuming a Poisson distribution of the noise, using this method one canestimate the number of false positive signals over the time course ofthe experiment. In some embodiments, the threshold level is calculatedas a value of 4 sigma above the background noise. For example, given anaverage background noise level of 200 photons, the analyzer systemestablishes a threshold level of 4√200 above the averagebackground/noise level of 200 photons to be 256 photons. Thus, in someembodiments, determining the concentration of a label in a sampleincludes establishing the threshold level above which photon signalsrepresent the presence of a label. Conversely, photon signals that havean energy level that is not greater than that of the threshold levelindicate the absence of a label.

Many bin measurements are taken to determine the concentration of asample, and the absence or presence of a label is ascertained for eachbin measurement. Typically, 60,000 measurements or more can made in oneminute (e.g., in embodiments in which the bin size is 1 ms—for smallerbin sizes the number of measurements is correspondingly larger, e.g.,6,000,000 measurements per minute for a bin size of 10 microseconds).Thus, no single measurement is crucial and the method provides for ahigh margin of error. The bins that are determined not to contain alabel (“no” bins) are discounted and only the measurements made in thebins that are determined to contain label (“yes” bins) are accounted indetermining the concentration of the label in the processing sample.Discounting measurements made in the “no” bins or bins that are devoidof label increases the signal to noise ratio and the accuracy of themeasurements. Thus, in some embodiments, determining the concentrationof a label in a sample comprises detecting the bin measurements thatreflect the presence of a label.

The signal to noise ratio or the sensitivity of the analyzer system canbe increased by minimizing the time that background noise is detectedduring a bin measurement in which a particle-label complex is detected.For example, in a bin measurement lasting 1 millisecond during which oneparticle-label complex is detected when passing across an interrogationspace within 250 microseconds, 750 microseconds of the 1 millisecond arespent detecting background noise emission. The signal to noise ratio canbe improved by decreasing the bin time. In some embodiments, the bintime is 1 millisecond. In other embodiments, the bin time is 750, 500,250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds or10 microseconds. Other bin times are as described herein.

Other factors that affect measurements are the brightness or dimness ofthe fluorescent moiety, the flow rate, and the power of the laser.Various combinations of the relevant factors that allow for detection oflabel will be apparent to those of skill in the art. In someembodiments, the bin time is adjusted without changing the flow rate. Itwill be appreciated by those of skill in the art that as bin timedecreases, laser power output directed at the interrogation space mustincrease to maintain a constant total energy applied to theinterrogation space during the bin time. For example, if bin time isdecreased from 1000 microseconds to 250 microseconds, as a firstapproximation, laser power output must be increased approximatelyfour-fold. These settings allow for the detection of the same number ofphotons in a 250 μs as the number of photons counted during the 1000 μsgiven the previous settings, and allow for faster analysis of samplewith lower backgrounds and thus greater sensitivity. In addition, flowrates may be adjusted in order to speed processing of sample. Thesenumbers are merely exemplary, and the skilled practitioner can adjustthe parameters as necessary to achieve the desired result.

In some embodiments, the interrogation space encompasses the entirecross-section of the sample stream. When the interrogation spaceencompasses the entire cross-section of the sample stream, only thenumber of labels counted and the volume passing through a cross-sectionof the sample stream in a set length of time are needed to calculate theconcentration of the label in the processing sample. In someembodiments, the interrogation space can be defined to be smaller thanthe cross-sectional area of sample stream by, for example, theinterrogation space is defined by the size of the spot illuminated bythe laser beam. In some embodiments, the interrogation space can bedefined by adjusting the apertures 306 (FIG. 1A) or 358 and 359 (FIG.1B) of the analyzer and reducing the illuminated volume that is imagedby the objective lens to the detector. In the embodiments when theinterrogation space is defined to be smaller than the cross-sectionalarea of sample stream, the concentration of the label can be determinedby interpolation of the signal emitted by the complex from a standardcurve that is generated using one or more samples of known standardconcentrations. In yet other embodiments, the concentration of the labelcan be determined by comparing the measured particles to an internallabel standard. In embodiments when a diluted sample is analyzed, thedilution factor is accounted in calculating the concentration of themolecule of interest in the starting sample.

As discussed above, when the interrogation space encompasses the entirecross-section of the sample stream, only the number of labels countedpassing through a cross-section of the sample stream in a set length oftime (bin) and the volume of sample that was interrogated in the bin areneeded to calculate the concentration the sample. The total number oflabels contained in the “yes” bins is determined and related to thesample volume represented by the total number of bins used in theanalysis to determine the concentration of labels in the processingsample. Thus, in one embodiment, determining the concentration of alabel in a processing sample comprises determining the total number oflabels detected “yes” bins and relating the total number of detectedlabels to the total sample volume that was analyzed. The total samplevolume that is analyzed is the sample volume that is passed through thecapillary flow cell and across the interrogation space in a specifiedtime interval. Alternatively, the concentration of the label complex ina sample is determined by interpolation of the signal emitted by thelabel in a number of bins from a standard curve that is generated bydetermining the signal emitted by labels in the same number of bins bystandard samples containing known concentrations of the label.

In some embodiments, the number of individual labels that are detectedin a bin is related to the relative concentration of the particle in theprocessing sample. At relatively low concentrations, for example atconcentrations below about 10⁻¹⁶ M the number of labels is proportionalto the photon signal that is detected in a bin. Thus, at lowconcentrations of label the photon signal is provided as a digitalsignal. At relatively higher concentrations, for example atconcentrations greater than about 10⁻¹⁶ M, the proportionality of photonsignal to a label is lost as the likelihood of two or more labelscrossing the interrogation space at about the same time and beingcounted as one becomes significant. Thus, in some embodiments,individual particles in a sample of a concentration greater than about10⁻¹⁶ M are resolved by decreasing the length of time of the binmeasurement.

Alternatively, in other embodiments, the total the photon signal that isemitted by a plurality of particles that are present in any one bin isdetected. These embodiments allow for single molecule detectors of theinvention wherein the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6,6.5, 7, 7.5, 8, or more than 8 logs.

“Dynamic range,” as that term is used herein, refers to the range ofsample concentrations that may be quantitated by the instrument withoutneed for dilution or other treatment to alter the concentration ofsuccessive samples of differing concentrations, where concentrations aredetermined with an accuracy appropriate for the intended use. Forexample, if a microliter plate contains a sample of 1 femtomolarconcentration for an analyte of interest in one well, a sample of 10,000femtomolar concentration for an analyte of interest in another well, anda sample of 100 femtomolar concentration for the analyte in a thirdwell, an instrument with a dynamic range of at least 4 logs and a lowerlimit of quantitation of 1 femtomolar is able to accurately quantitatethe concentration of all the samples without the need for furthertreatment to adjust concentration, e.g., dilution. Accuracy may bedetermined by standard methods, e.g., using a series of standards ofconcentrations that span the dynamic range and constructing a standardcurve. Standard measures of fit of the resulting standard curve may beused as a measure of accuracy, e.g., an r² greater than about 0.7, 0.75,0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

Increased dynamic range is achieved by altering the manner in which datafrom the detector is analyzed, and/or by the use of an attenuatorbetween the detector and the interrogation space. At the low end of therange, where processing sample is sufficiently dilute that eachdetection event, i.e., each burst of photons above a threshold level ina bin (the “event photons”), likely represents only one label, the datais analyzed to count detection events as single molecules. I.e., eachbin is analyzed as a simple “yes” or “no” for the presence of label, asdescribed above. For a more concentrated processing sample, where thelikelihood of two or more labels occupying a single bin becomessignificant, the number of event photons in a significant number of binsis found to be substantially greater than the number expected for asingle label, e.g., the number of event photons in a significant numberof bins corresponds to two-fold, three-fold, or more, than the number ofevent photons expected for a single label. For these samples, theinstrument changes its method of data analysis to one of integrating thetotal number of event photons for the bins of the processing sample.This total will be proportional to the total number of labels that werein all the bins. For an even more concentrated processing sample, wheremany labels are present in most bins, background noise becomes aninsignificant portion of the total signal from each bin, and theinstrument changes its method of data analysis to one of counting totalphotons per bin (including background). An even further increase indynamic range can be achieved by the use of an attenuator between theflow cell and the detector, when concentrations are such that theintensity of light reaching the detector would otherwise exceed thecapacity of the detector for accurately counting photons, i.e., saturatethe detector.

The instrument may include a data analysis system that receives inputfrom the detector and determines the appropriate analysis method for thesample being run, and outputs values based on such analysis. The dataanalysis system may further output instructions to use or not use anattenuator, if an attenuator is included in the instrument.

By utilizing such methods, the dynamic range of the instrument can bedramatically increased. Thus, in some embodiments, the instrument iscapable of measuring concentrations of samples over a dynamic range ofmore than about 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000(5.5 log), 1,000,000 (6 log), 3,500,000 (6.5 log), 10,000,000 (7 log),35,000,000 (7.5 log), or 100,000,000 (8 log). In some embodiments, theinstrument is capable of measuring concentrations of samples over adynamic range of more than about 100,000 (5 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 1,000,000 (6 log). In some embodiments,the instrument is capable of measuring concentrations of samples over adynamic range of more than about 10,000,000 (7 log). In someembodiments, the instrument is capable of measuring the concentrationsof samples over a dynamic range of from about 1-10 femtomolar to atleast about 1000; 10,000; 100,000; 350,000; 1,000,000; 3,500,000;10,000,000, or 35,000,000 femtomolar. In some embodiments, theinstrument is capable of measuring the concentrations of samples over adynamic range of from about 1-10 femtomolar to at least about 10,000femtomolar. In some embodiments, the instrument is capable of measuringthe concentrations of samples over a dynamic range of from about 1-10femtomolar to at least about 100,000 femtomolar. In some embodiments,the instrument is capable of measuring the concentrations of samplesover a dynamic range of from about 1-10 femtomolar to at least about1,000,000 femtomolar. In some embodiments, the instrument is capable ofmeasuring the concentrations of samples over a dynamic range of fromabout 1-10 femtomolar to at least about 10,000,000.

In some embodiments, an analyzer or analyzer system of the invention iscapable of detecting an analyte, e.g., a biomarker at a limit ofdetection of less than 1 nanomolar, or 1 picomolar, or 1 femtomolar, or1 attomolar, or 1 zeptomolar. In some embodiments, the analyzer oranalyzer system is capable of detecting a change in concentration of theanalyte, or of multiple analytes, e.g., a biomarker or biomarkers, fromone sample to another sample of less than about 0.1, 1, 2, 5, 10, 20,30, 40, 50, 60, or 80% when the biomarker is present at a concentrationof less than 1 nanomolar, or 1 picomolar, or 1 femtomolar, or 1attomolar, or 1 zeptomolar, in the samples, and when the size of each ofthe sample is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1,0.01, 0.001, or 0.0001 ul. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 1 picomolar,and when the size of each of the samples is less than about 50 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 100 femtomolar, and when the sizeof each of the samples is less than about 50 μl. In some embodiments,the analyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 50 femtomolar, and when the size of each of the samplesis less than about 50 μl. In some embodiments, the analyzer or analyzersystem is capable of detecting a change in concentration of the analytefrom a first sample to a second sample of less than about 20%, when theanalyte is present at a concentration of less than about 5 femtomolar,and when the size of each of the samples is less than about 50 μl. Insome embodiments, the analyzer or analyzer system is capable ofdetecting a change in concentration of the analyte from a first sampleto a second sample of less than about 20%, when the analyte is presentat a concentration of less than about 5 femtomolar, and when the size ofeach of the samples is less than about 5 μl. In some embodiments, theanalyzer or analyzer system is capable of detecting a change inconcentration of the analyte from a first sample to a second sample ofless than about 20%, when the analyte is present at a concentration ofless than about 1 femtomolar, and when the size of each of the samplesis less than about 5 μl.

V. Instruments and Systems Suitable for Highly Sensitive Analysis ofMolecules

The methods of the invention utilize analytical instruments of highsensitivity, e.g., single molecule detectors. Such single moleculedetectors include embodiments as hereinafter described.

In some embodiments, the invention provides an analyzer system kit fordetecting a single protein molecule in a sample, said system includes ananalyzer system for detecting a single protein molecule in a sample andleast one label that includes a fluorescent moiety and a binding partnerfor the protein molecule, where the analyzer includes an electromagneticradiation source for stimulating the fluorescent moiety; a capillaryflow cell for passing the label; a source of motive force for moving thelabel in the capillary flow cell; an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source; and an electromagnetic radiationdetector operably connected to the interrogation space for measuring anelectromagnetic characteristic of the stimulated fluorescent moiety,where the fluorescent moiety is capable of emitting at least about 200photons when simulated by a laser emitting light at the excitationwavelength of the moiety, where the laser is focused on a spot not lessthan about 5 microns in diameter that contains the moiety, and where thetotal energy directed at the spot by the laser is no more than about 3microJoules.

One embodiment of an analyzer kit of the invention is depicted in FIG.12. The kit includes a label for a protein molecule that includes abinding partner for a protein molecule and a fluorescent moiety. The kitfurther includes an analyzer system for detecting a single proteinmolecule (300) that includes an electromagnetic radiation source 301 forstimulating the fluorescent moiety, a capillary flow cell 313 forpassing the label; a source of motive force for moving the label in thecapillary flow cell (not shown); an interrogation space defined withinthe capillary flow cell for receiving electromagnetic radiation emittedfrom the electromagnetic source 314 (FIG. 2A); and an electromagneticradiation detector 309 operably connected to the interrogation space formeasuring an electromagnetic characteristic of the stimulatedfluorescent moiety, where the fluorescent moiety is capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, where the laser is focused on aspot not less than about 5 microns in diameter that contains the moiety,and where the total energy directed at the spot by the laser is no morethan about 3 microJoules. In some embodiments, the beam 311 from anelectromagnetic radiation source 301 is focused by the microscopeobjective 315 to form one interrogation space 314 (FIG. 2A) within thecapillary flow cell 313. the microscope objective may have a numericalaperture of equal to or greater than 0.7, 0.8, 0.9, or 1.0 in someembodiments.

In some embodiments of the analyzer system kit, the analyzer comprisesnot more than one interrogation space. In some embodiments, theelectromagnetic radiation source is a laser that has a power output ofat least about 3, 5, 10, or 20 mW. In some embodiments, the fluorescentmoiety comprises a fluorescent molecule. In some embodiments, thefluorescent molecule is a dye molecule, such as a dye molecule thatcomprises at least one substituted indolium ring system in which thesubstituent on the 3-carbon of the indolium ring contains a chemicallyreactive group or a conjugated substance. In some embodiments, thefluorescent moiety is a quantum dot. In some embodiments, theelectromagnetic radiation source is a continuous wave electromagneticradiation source, such as a light-emitting diode or a continuous wavelaser. In some embodiments, the motive force is pressure. In someembodiments, the detector is an avalanche photodiode detector. In someembodiments, the analyzer utilizes a confocal optical arrangement fordeflecting a laser beam onto said interrogation space and for imagingsaid stimulated dye molecule (shown in FIG. 12), wherein said confocaloptical arrangement comprises an objective lens having a numericalaperture of at least about 0.8. In some embodiments, the analyzerfurther comprises a sampling system capable of automatically sampling aplurality of samples and providing a fluid communication between asample container and said interrogation space. In some embodiments, theanalyzer system further comprises a sample recovery system in fluidcommunication with said interrogation space, wherein said recoverysystem is capable of recovering substantially all of said sample. Insome embodiments, the kit further includes instructions for use of thesystem.

A. Apparatus/System

In one aspect, the methods described herein utilize an analyzer systemcapable of detecting a single molecule in a sample. In one embodiment,the analyzer system is capable of single molecule detection of afluorescently labeled particle wherein the analyzer system detectsenergy emitted by an excited fluorescent label in response to exposureby an electromagnetic radiation source when the single particle ispresent in an interrogation space defined within a capillary flow cellfluidly connected to the sampling system of the analyzer system. In afurther embodiment of the analyzer system, the single particle movesthrough the interrogation space of the capillary flow cell by means of amotive force. In another embodiment of the analyzer system, an automaticsampling system may be included in the analyzer system for introducingthe sample into the analyzer system. In another embodiment of theanalyzer system, a sample preparation system may be included in theanalyzer system for preparing a sample. In a further embodiment, theanalyzer system may contain a sample recovery system for recovering atleast a portion of the sample after analysis is complete.

In one aspect, the analyzer system consists of an electromagneticradiation source for exciting a single particle labeled with afluorescent label. In one embodiment, the electromagnetic radiationsource of the analyzer system is a laser. In a further embodiment, theelectromagnetic radiation source is a continuous wave laser.

In a typical embodiment, the electromagnetic radiation source excites afluorescent moiety attached to a label as the label passes through theinterrogation space of the capillary flow cell. In some embodiments, thefluorescent label moiety includes one or more fluorescent dye molecules.In some embodiments, the fluorescent label moiety is a quantum dot. Anyfluorescent moiety as described herein may be used in the label.

A label is exposed to electromagnetic radiation when the label passesthrough an interrogation space located within the capillary flow cell.The interrogation space is typically fluidly connected to a samplingsystem. In some embodiments the label passes through the interrogationspace of the capillary flow cell due to a motive force to advance thelabel through the analyzer system. The interrogation space is positionedsuch that it receives electromagnetic radiation emitted from theradiation source. In some embodiments, the sampling system is anautomated sampling system capable of sampling a plurality of sampleswithout intervention from a human operator.

The label passes through the interrogation space and emits a detectableamount of energy when excited by the electromagnetic radiation source.In one embodiment, an electromagnetic radiation detector is operablyconnected to the interrogation space. The electromagnetic radiationdetector is capable of detecting the energy emitted by the label, e.g.,by the fluorescent moiety of the label.

In a further embodiment of the analyzer system, the system furtherincludes a sample preparation mechanism where a sample may be partiallyor completely prepared for analysis by the analyzer system. In someembodiments of the analyzer system, the sample is discarded after it isanalyzed by the system. In other embodiments, the analyzer systemfurther includes a sample recovery mechanism whereby at least a portion,or alternatively all or substantially all, of the sample may berecovered after analysis. In such an embodiment, the sample can bereturned to the origin of the sample. In some embodiments, the samplecan be returned to microtiter wells on a sample microtiter plate. Theanalyzer system typically further consists of a data acquisition systemfor collecting and reporting the detected signal.

B. Single Particle Analyzer

As shown in FIG. 1A, described herein is one embodiment of an analyzersystem 300. The analyzer system 300 includes an electromagneticradiation source 301, a mirror 302, a lens 303, a capillary flow cell313, a microscopic objective lens 305, an aperture 306, a detector lens307, a detector filter 308, a single photon detector 309, and aprocessor 310 operatively connected to the detector.

In operation the electromagnetic radiation source 301 is aligned so thatits output 311 is reflected off of a front surface 312 of mirror 302.The lens 303 focuses the beam 311 onto a single interrogation space (anillustrative example of an interrogation space 314 is shown in FIG. 2A)in the capillary flow cell 313. The microscope objective lens 305collects light from sample particles and forms images of the beam ontothe aperture 306. The aperture 306 affects the fraction of light emittedby the specimen in the interrogation space of the capillary flow cell313 that can be collected. The detector lens 307 collects the lightpassing through the aperture 306 and focuses the light onto an activearea of the detector 309 after it passes through the detector filters308. The detector filters 308 minimize aberrant noise signals due tolight scatter or ambient light while maximizing the signal emitted bythe excited fluorescent moiety bound to the particle. The processor 310processes the light signal from the particle according to the methodsdescribed herein.

In one embodiment, the microscope objective lens 305 is a high numericalaperture microscope objective. As used herein, “high numerical aperturelens” include a lens with a numerical aperture of equal to or greaterthan 0.6. The numerical aperture is a measure of the number of highlydiffracted image-forming light rays captured by the objective. A highernumerical aperture allows increasingly oblique rays to enter theobjective lens and thereby produce a more highly resolved image.Additionally, the brightness of an image increases with a highernumerical aperture. High numerical aperture lenses are commerciallyavailable from a variety of vendors, and any one lens having a numericalaperture of equal to or greater than approximately 0.6 may be used inthe analyzer system. In some embodiments, the lens has a numericalaperture of about 0.6 to about 1.3. In some embodiments, the lens has anumerical aperture of about 0.6 to about 1.0. In some embodiments, thelens has a numerical aperture of about 0.7 to about 1.2. In someembodiments, the lens has a numerical aperture of about 0.7 to about1.0. In some embodiments, the lens has a numerical aperture of about 0.7to about 0.9. In some embodiments, the lens has a numerical aperture ofabout 0.8 to about 1.3. In some embodiments, the lens has a numericalaperture of about 0.8 to about 1.2. In some embodiments, the lens has anumerical aperture of about 0.8 to about 1.0. In some embodiments, thelens has a numerical aperture of at least about 0.6. In someembodiments, the lens has a numerical aperture of at least about 0.7. Insome embodiments, the lens has a numerical aperture of at least about0.8. In some embodiments, the lens has a numerical aperture of at leastabout 0.9. In some embodiments, the lens has a numerical aperture of atleast about 1.0. In some embodiments, the aperture of the microscopeobjective lens 305 is approximately 1.25. In an embodiment where amicroscope objective lens 305 of 0.8 is used, a Nikon 60×/0.8 NAAchromat lens (Nikon, Inc., USA) can be used.

In some embodiments, the electromagnetic radiation source 301 is a laserthat emits light in the visible spectrum. In all embodiments, theelectromagnetic radiation source is set such that wavelength of thelaser is set such that it is of a sufficient wavelength to excite thefluorescent label attached to the particle. In some embodiments, thelaser is a continuous wave laser with a wavelength of 639 nm. In otherembodiments, the laser is a continuous wave laser with a wavelength of532 nm. In other embodiments, the laser is a continuous wave laser witha wavelength of 422 nm. In other embodiments, the laser is a continuouswave laser with a wavelength of 405 nm. Any continuous wave laser with awavelength suitable for exciting a fluorescent moiety as used in themethods and compositions of the invention may be used without departingfrom the scope of the invention.

In a single particle analyzer system 300, as each particle passesthrough the beam 311 of the electromagnetic radiation source, theparticle enters into an excited state. When the particle relaxes fromits excited state, a detectable burst of light is emitted. Theexcitation-emission cycle is repeated many times by each particle in thelength of time it takes for it to pass through the beam allowing theanalyzer system 300 to detect tens to thousands of photons for eachparticle as it passes through an interrogation space 314. Photonsemitted by fluorescent particles are registered by the detector 309(FIG. 1A) with a time delay indicative of the time for the particlelabel complex to pass through the interrogation space. The photonintensity is recorded by the detector 309 and sampling time is dividedinto bins, which are uniform, arbitrary, time segments with freelyselectable time channel widths. The number of signals contained in eachbin evaluated. One or a combination of several statistical analyticalmethods are employed in order to determine when a particle is present.Such methods include determining the baseline noise of the analyzersystem and setting a signal strength for the fluorescent label at astatistical level above baseline noise to eliminate false positivesignals from the detector.

The electromagnetic radiation source 301 is focused onto a capillaryflow cell 313 of the analyzer system 300 where the capillary flow cell313 is fluidly connected to the sample system. An interrogation space314 is shown in FIG. 2A. The beam 311 from the continuous waveelectromagnetic radiation source 301 of FIG. 1A is optically focused toa specified depth within the capillary flow cell 313. The beam 311 isdirected toward the sample-filled capillary flow cell 313 at an angleperpendicular to the capillary flow cell 313. The beam 311 is operatedat a predetermined wavelength that is selected to excite a particularfluorescent label used to label the particle of interest. The size orvolume of the interrogation space 314 is determined by the diameter ofthe beam 311 together with the depth at which the beam 311 is focused.Alternatively, the interrogation space can be determined by running acalibration sample of known concentration through the analyzer system.

When single molecules are detected in the sample concentration, the beamsize and the depth of focus required for single molecule detection areset and thereby define the size of the interrogation space 314. Theinterrogation space 314 is set such that, with an appropriate sampleconcentration, only one particle is present in the interrogation space314 during each time interval over which time observations are made.

It will be appreciated that the detection interrogation volume asdefined by the beam is not perfectly spherically shaped, and typicallyis a “bow-tie” shape. However, for the purposes of definition, “volumes”of interrogation spaces are defined herein as the volume encompassed bya sphere of a diameter equal to the focused spot diameter of the beam.The focused spot of the beam 311 may have various diameters withoutdeparting from the scope of the present invention. In some embodiments,the diameter of the focused spot of the beam is about 1 to about 5, 10,15, or 20 microns, or about 5 to about 10, 15, or 20 microns, or about10 to about 20 microns, or about 10 to about 15 microns. In someembodiments, the diameter of the focused spot of the beam is about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20microns. In some embodiments, the diameter of the focused spot of thebeam is about 5 microns. In some embodiments, the diameter of thefocused spot of the beam is about 10 microns. In some embodiments, thediameter of the focused spot of the beam is about 12 microns. In someembodiments, the diameter of the focused spot of the beam is about 13microns. In some embodiments, the diameter of the focused spot of thebeam is about 14 microns. In some embodiments, the diameter of thefocused spot of the beam is about 15 microns. In some embodiments, thediameter of the focused spot of the beam is about 16 microns. In someembodiments, the diameter of the focused spot of the beam is about 17microns. In some embodiments, the diameter of the focused spot of thebeam is about 18 microns. In some embodiments, the diameter of thefocused spot of the beam is about 19 microns. In some embodiments, thediameter of the focused spot of the beam is about 20 microns.

In an alternate embodiment of the single particle analyzer system, morethan one electromagnetic radiation source can be used to exciteparticles labeled with fluorescent labels of different wavelengths. Inanother alternate embodiment, more than one interrogation space in thecapillary flow cell can be used. In another alternate embodiment,multiple detectors can be employed to detect different emissionwavelengths from the fluorescent labels. An illustration incorporatingeach of these alternative embodiments of an analyzer system is shown inFIG. 1B. These embodiments are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660.

In some embodiments of the analyzer system 300, a motive force isrequired to move a particle through the capillary flow cell 313 of theanalyzer system 300. In one embodiment, the motive force can be a formof pressure. The pressure used to move a particle through the capillaryflow cell can be generated by a pump. In some embodiments, a Scivex,Inc. HPLC pump can be used. In some embodiments where a pump is used asa motive force, the sample can pass through the capillary flow cell at arate of 1 μL/min to about 20 μL/min, or about 5 μL/min to about 20μL/min. In some embodiments, the sample can pass through the capillaryflow cell at a rate of about 5 μL/min. In some embodiments, the samplecan pass through the capillary flow cell at a rate of about 10 μL/min.In some embodiments, the sample can pass through the capillary flow cellat a rate of about 15 μL/min. In some embodiments, the sample can passthrough the capillary flow cell at a rate of about 20 μL/min. In someembodiments, an electrokinetic force can be used to move the particlethrough the analyzer system. Such a method has been previously disclosedand is incorporated by reference from previous U.S. patent applicationSer. No. 11/048,660.

In one aspect of the analyzer system 300, the detector 309 of theanalyzer system detects the photons emitted by the fluorescent label. Inone embodiment, the photon detector is a photodiode. In a furtherembodiment, the detector is an avalanche photodiode detector. In someembodiments, the photodiodes can be silicon photodiodes with awavelength detection of 190 nm and 1100 nm. When germanium photodiodesare used, the wavelength of light detected is between 400 nm to 1700 nm.In other embodiments, when an indium gallium arsenide photodiode isused, the wavelength of light detected by the photodiode is between 800nm and 2600 nm. When lead sulfide photodiodes are used as detectors, thewavelength of light detected is between 1000 nm and 3500 nm.

In some embodiments, the optics of the electromagnetic radiation source301 and the optics of the detector 309 are arranged in a conventionaloptical arrangement. In such an arrangement, the electromagneticradiation source and the detector are aligned on different focal planes.The arrangement of the laser and the detector optics of the analyzersystem as shown in FIGS. 1A and 1B is that of a conventional opticalarrangement.

In some embodiments, the optics of the electromagnetic radiation sourceand the optics of the detector are arranged in a confocal opticalarrangement. In such an arrangement, the electromagnetic radiationsource 301 and the detector 309 are aligned on the same focal plane. Theconfocal arrangement renders the analyzer more robust because theelectromagnetic radiation source 301 and the detector optics 309 do notneed to be realigned if the analyzer system is moved. This arrangementalso makes the use of the analyzer more simplified because it eliminatesthe need to realign the components of the analyzer system. The confocalarrangement for the analyzer 300 (FIG. 1A) and the analyzer 355 (FIG.1B) are shown in FIGS. 3A and 3B respectively. FIG. 3A shows that thebeam 311 from an electromagnetic radiation source 301 is focused by themicroscope objective 315 to form one interrogation space 314 (FIG. 2A)within the capillary flow cell 313. A dichroic mirror 316, whichreflects laser light but passes fluorescent light, is used to separatethe fluorescent light from the laser light. Filter 317 that ispositioned in front of the detector eliminates any non-fluorescent lightat the detector. In some embodiments, an analyzer system configured in aconfocal arrangement can comprise two or more interrogations spaces.Such a method has been previously disclosed and is incorporated byreference from previous U.S. patent application Ser. No. 11/048,660.

The laser can be a tunable dye laser, such as a helium-neon laser. Thelaser can be set to emit a wavelength of 632.8 nm. Alternatively, thewavelength of the laser can be set to emit a wavelength of 543.5 nm or1523 nm. Alternatively, the electromagnetic laser can be an argon ionlaser. In such an embodiment, the argon ion laser can be operated as acontinuous gas laser at about 25 different wavelengths in the visiblespectrum, the wavelength set between 408.9 and 686.1 nm but at itsoptimum performance set between 488 and 514.5 nm.

1 Electromagnetic Radiation Source

In some embodiments of the analyzer system a chemiluminescent label maybe used. In such an embodiment, it may not be necessary to utilize an EMsource for detection of the particle. In another embodiment, theextrinsic label or intrinsic characteristic of the particle is alight-interacting label or characteristic, such as a fluorescent labelor a light-scattering label. In such an embodiment, a source of EMradiation is used to illuminate the label and/or the particle. EMradiation sources for excitation of fluorescent labels are preferred.

In some embodiments, the analyzer system consists of an electromagneticradiation source 301. Any number of radiation sources may be used in anyone analyzer system 300 without departing from the scope of theinvention. Multiple sources of electromagnetic radiation have beenpreviously disclosed and are incorporated by reference from previousU.S. patent application Ser. No. 11/048,660. In some embodiments, allthe continuous wave electromagnetic (EM) radiation sources emitelectromagnetic radiation at the same wavelengths. In other embodiments,different sources emit different wavelengths of EM radiation.

In one embodiment, the EM source(s) 301, 351, 352 are continuous wavelasers producing wavelengths of between 200 nm and 1000 nm. Such EMsources have the advantage of being small, durable and relativelyinexpensive. In addition, they generally have the capacity to generatelarger fluorescent signals than other light sources. Specific examplesof suitable continuous wave EM sources include, but are not limited to:lasers of the argon, krypton, helium-neon, helium-cadmium types, as wellas, tunable diode lasers (red to infrared regions), each with thepossibility of frequency doubling. The lasers provide continuousillumination with no accessory electronic or mechanical devices, such asshutters, to interrupt their illumination. In an embodiment where acontinuous wave laser is used, an electromagnetic radiation source of 3mW may be of sufficient energy to excite a fluorescent label. A beamfrom a continuous wave laser of such energy output may be between 2 to 5μm in diameter. The time of exposure of the particle to laser beam inorder to be exposed to 3 mW may be a time period of about 1 msec. Inalternate embodiments, the time of exposure to the laser beam may beequal to or less than about 500 μsec. In an alternate embodiment, thetime of exposure may be equal to or less than about 100 μsec. In analternate embodiment, the time of exposure may be equal to or less thanabout 50 μsec. In an alternate embodiment, the time of exposure may beequal to or less than about 10 μsec.

LEDs are another low-cost, high reliability illumination source. Recentadvances in ultra-bright LEDs and dyes with high absorptioncross-section and quantum yield support the applicability of LEDs tosingle particle detection. Such lasers could be used alone or incombination with other light sources such as mercury arc lamps,elemental arc lamps, halogen lamps, arc discharges, plasma discharges,light-emitting diodes, or combination of these.

In other embodiments, the EM source could be in the form of a pulse wavelaser. In such an embodiment, the pulse size of the laser is animportant factor. In such an embodiment, the size, focus spot, and thetotal energy emitted by the laser is important and must be of sufficientenergy as to be able to excite the fluorescent label. When a pulse laseris used, a pulse of longer duration may be required. In some embodimentsa laser pulse of 2 nanoseconds may be used. In some embodiments a laserpulse of 5 nanoseconds may be used. In some embodiments a pulse ofbetween 2 to 5 nanoseconds may be used.

The optimal laser intensity depends on the photo bleachingcharacteristics of the single dyes and the length of time required totraverse the interrogation space (including the speed of the particle,the distance between interrogation spaces if more than one is used andthe size of the interrogation space(s)). To obtain a maximal signal, itis desirable to illuminate the sample at the highest intensity whichwill not result in photo bleaching a high percentage of the dyes. Thepreferred intensity is one such that no more that 5% of the dyes arebleached by the time the particle has traversed the interrogation space.

The power of the laser is set depending on the type of dye moleculesthat need to be stimulated and the length of time the dye molecules arestimulated, and/or the speed with which the dye molecules pass throughthe capillary flow cell. Laser power is defined as the rate at whichenergy is delivered by the beam and is measured in units ofJoules/second, or Watts. It will be appreciated that the greater thepower output of the laser, the shorter the time that the laserilluminates the particle may be, while providing a constant amount ofenergy to the interrogation space while the particle is passing throughthe space. Thus, in some embodiments, the combination of laser power andtime of illumination is such that the total energy received by theinterrogation space during the time of illumination is more than about0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 microJoule. In some embodiments, thecombination of laser power and time of illumination is such that thetotal energy received by the interrogation space during the time ofillumination is less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoule.In some embodiments, the combination of laser power and time ofillumination is such that the total energy received by the interrogationspace during the time of illumination is between about 0.1 and 100microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 100 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 1and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 2and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 60 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 50 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 40 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is between about 3and 30 microJoule. In some embodiments, the combination of laser powerand time of illumination is such that the total energy received by theinterrogation space during the time of illumination is about 1microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 3microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 5microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 10microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 15microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 20microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 30microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 40microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 50microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 60microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 70microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 80microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 90microJoule. In some embodiments, the combination of laser power and timeof illumination is such that the total energy received by theinterrogation space during the time of illumination is about 100microJoule.

In some embodiments, the laser power output is set to at least about 1mW, 2 mW, 3 mW, 4 mW, 5 mW, 6, mw, 7 mW, 8 mW, 9 mW, 10 mW, 13 mW, 15mW, 20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70 mW, 80 mW, 90 mW, 100mW, or more than 100 mW. In some embodiments, the laser power output isset to at least about 1 mW. In some embodiments, the laser power outputis set to at least about 3 mW. In some embodiments, the laser poweroutput is set to at least about 5 mW. In some embodiments, the laserpower output is set to at least about 10 mW. In some embodiments, thelaser power output is set to at least about 15 mW. In some embodiments,the laser power output is set to at least about 20 mW. In someembodiments, the laser power output is set to at least about 30 mW. Insome embodiments, the laser power output is set to at least about 40 mW.In some embodiments, the laser power output is set to at least about 50mW. In some embodiments, the laser power output is set to at least about60 mW. In some embodiments, the laser power output is set to at leastabout 90 mW.

The time that the laser illuminates the interrogation space can be setto no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, or1000 microseconds. The time that the laser illuminates the interrogationspace can be set to no more than about 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600,700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laserilluminates the interrogation space can be set between about 1 and 1000microseconds. The time that the laser illuminates the interrogationspace can be set between about 5 and 500 microseconds. The time that thelaser illuminates the interrogation space can be set between about 5 and100 microseconds. The time that the laser illuminates the interrogationspace can be set between about 10 and 100 microseconds. The time thatthe laser illuminates the interrogation space can be set between about10 and 50 microseconds. The time that the laser illuminates theinterrogation space can be set between about 10 and 20 microseconds. Thetime that the laser illuminates the interrogation space can be setbetween about 5 and 50 microseconds. The time that the laser illuminatesthe interrogation space can be set between about 1 and 100 microseconds.In some embodiments, the time that the laser illuminates theinterrogation space is about 1 microsecond. In some embodiments, thetime that the laser illuminates the interrogation space is about 5microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 10 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 25microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 50 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 100microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 250 microseconds. In some embodiments,the time that the laser illuminates the interrogation space is about 500microseconds. In some embodiments, the time that the laser illuminatesthe interrogation space is about 1000 microseconds.

For example, the time that the laser illuminates the interrogation spacecan be set to 1 millisecond, 250 microseconds, 100 microseconds, 50microseconds, 25 microseconds or 10 microseconds with a laser thatprovides a power output of 3 mW, 4 mw, 5 mW, or more than 5 mW. In someembodiments, a label is illuminated with a laser that provides a poweroutput of 3 mW and illuminates the label for about 1000 microseconds. Inother embodiments, a label is illuminated for less than 1000milliseconds with a laser providing a power output of not more thanabout 20 mW. In other embodiments, the label is illuminated with a laserpower output of 20 mW for less than or equal to about 250 microseconds.In some embodiments, the label is illuminated with a laser power outputof about 5 mW for less than or equal to about 1000 microseconds.

2. Capillary Flow Cell

The capillary flow cell is fluidly connected to the sample system. Inone embodiment, the interrogation space 314 of an analyzer system, isdetermined by the cross sectional area of the corresponding beam 311 andby a segment of the beam within the field of view of the detector 309.In one embodiment of the analyzer system, the interrogation space 314has a volume, as defined herein, of between about between about 0.01 and500 pL, or between about 0.01 pL and 100 pL, or between about 0.01 pLand 10 pL, or between about 0.01 pL and 1 pL, or between about 0.01 pLand 0.5 pL, or between about 0.02 pL and about 300 pL, or between about0.02 pL and about 50 pL or between about 0.02 pL and about 5 pL orbetween about 0.02 pL and about 0.5 pL or between about 0.02 pL andabout 2 pL, or between about 0.05 pL and about 50 pL, or between about0.05 pL and about 5 pL, or between about 0.05 pL and about 0.5 pL, orbetween about 0.05 pL and about 0.2 pL, or between about 0.1 pL andabout 25 pL. In some embodiments, the interrogation space has a volumebetween about 0.01 pL and 10 pL. In some embodiments, the interrogationspace 314 has a volume between about 0.01 pL and 1 pL. In someembodiments, the interrogation space 314 has a volume between about 0.02pL and about 5 pL. In some embodiments, the interrogation space 314 hasa volume between about 0.02 pL and about 0.5 pL. In some embodiments,the interrogation space 314 has a volume between about 0.05 pL and about0.2 pL. In some embodiments, the interrogation space 314 has a volume ofabout 0.1 pL. Other useful interrogation space volumes are as describedherein. It should be understood by one skilled in the art that theinterrogation space 314 can be selected for maximum performance of theanalyzer. Although very small interrogation spaces have been shown tominimize the background noise, large interrogation spaces have theadvantage that low concentration samples can be analyzed in a reasonableamount of time. In embodiments in which two interrogation spaces 370 and371 are used, volumes such as those described herein for a singleinterrogation space 314 may be used.

In one embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 femtomolar (fM) to about 1 zeptomolar (zM). Inone embodiment of the present invention, the interrogation spaces arelarge enough to allow for detection of particles at concentrationsranging from about 1000 fM to about 1 attomolar (aM). In one embodimentof the present invention, the interrogation spaces are large enough toallow for detection of particles at concentrations ranging from about 10fM to about 1 attomolar (aM). In many cases, the large interrogationspaces allow for the detection of particles at concentrations of lessthan about 1 fM without additional pre-concentration devices ortechniques. One skilled in the art will recognize that the mostappropriate interrogation space size depends on the brightness of theparticles to be detected, the level of background signal, and theconcentration of the sample to be analyzed.

The size of the interrogation space 314 can be limited by adjusting theoptics of the analyzer. In one embodiment, the diameter of the beam 311can be adjusted to vary the volume of the interrogation space 314. Inanother embodiment, the field of view of the detector 309 can be varied.Thus, the source 301 and the detector 309 can be adjusted so that singleparticles will be illuminated and detected within the interrogationspace 314. In another embodiment, the width of aperture 306 (FIG. 1A)that determine the field of view of the detector 309 is variable. Thisconfiguration allows for altering the interrogation space, in near realtime, to compensate for more or less concentrated samples, ensuring alow probability of two or more particles simultaneously being within aninterrogation space. Similar alterations for two or more interrogationspaces, 370 and 371, may performed.

In another embodiment, the interrogation space can be defined throughthe use of a calibration sample of known concentration that is passedthrough the capillary flow cell prior to the actual sample being tested.When only one single particle is detected at a time in the calibrationsample as the sample is passing through the capillary flow cell, thedepth of focus together with the diameter of the beam of theelectromagnetic radiation source determines the size of theinterrogation space in the capillary flow cell.

Physical constraints to the interrogation spaces can also be provided bya solid wall. In one embodiment, the wall is one or more of the walls ofa flow cell 313 (FIG. 2A), when the sample fluid is contained within acapillary. In one embodiment, the cell is made of glass, but othersubstances transparent to light in the range of about 200 to about 1,000nm or higher, such as quartz, fused silica, and organic materials suchas Teflon, nylon, plastics, such as polyvinylchloride, polystyrene, andpolyethylene, or any combination thereof, may be used without departingfrom the scope of the present invention. Although other cross-sectionalshapes (e.g., rectangular, cylindrical) may be used without departingfrom the scope of the present invention, in one embodiment the capillaryflow cell 313 has a square cross section. In another embodiment, theinterrogation space may be defined at least in part by a channel (notshown) etched into a chip (not shown). Similar considerations apply toembodiments in which two interrogation spaces are used (370 and 371 inFIG. 2B).

The interrogation space is bathed in a fluid. In one embodiment, thefluid is aqueous. In other embodiments, the fluid is non-aqueous or acombination of aqueous and non-aqueous fluids. In addition the fluid maycontain agents to adjust pH, ionic composition, or sieving agents, suchas soluble macroparticles or polymers or gels. It is contemplated thatvalves or other devices may be present between the interrogation spacesto temporarily disrupt the fluid connection. Interrogation spacestemporarily disrupted are considered to be connected by fluid.

In another embodiment of the invention, an interrogation space is thesingle interrogation space present within the flow cell 313 which isconstrained by the size of a laminar flow of the sample material withina diluent volume, also called sheath flow. In these and otherembodiments, the interrogation space can be defined by sheath flow aloneor in combination with the dimensions of the illumination source or thefield of view of the detector. Sheath flow can be configured in numerousways, including: The sample material is the interior material in aconcentric laminar flow, with the diluent volume in the exterior; thediluent volume is on one side of the sample volume; the diluent volumeis on two sides of the sample material; the diluent volume is onmultiple sides of the sample material, but not enclosing the samplematerial completely; the diluent volume completely surrounds the samplematerial; the diluent volume completely surrounds the sample materialconcentrically; the sample material is the interior material in adiscontinuous series of drops and the diluent volume completelysurrounds each drop of sample material.

In some embodiments, single molecule detectors of the invention compriseno more than one interrogation space. In some embodiments, multipleinterrogation spaces are used. Multiple interrogation spaces have beenpreviously disclosed and are incorporated by reference from U.S. patentapplication Ser. No. 11/048,660. One skilled in the art will recognizethat in some cases the analyzer will contain 2, 3, 4, 5, 6 or moredistinct interrogation spaces.

3. Motive Force

In one embodiment of the analyzer system, the particles are movedthrough the interrogation space by a motive force. In some embodiments,the motive force for moving particles is pressure. In some embodiments,the pressure is supplied by a pump, and air pressure source, a vacuumsource, a centrifuge, or a combination thereof. In some embodiments, themotive force for moving particles is an electrokinetic force. The use ofan electrokinetic force as a motive force has been previously disclosedin a prior application and is incorporated by reference from U.S. patentapplication Ser. No. 11/048,660.

In one embodiment, pressure can be used as a motive force to moveparticles through the interrogation space of the capillary flow cell. Ina further embodiment, pressure is supplied to move the sample by meansof a pump. Suitable pumps are known in the art. In one embodiment, pumpsmanufactured for HPLC applications, such as those made by Scivax, Inc.can be used as a motive force. In other embodiments, pumps manufacturedfor microfluidics applications can be used when smaller volumes ofsample are being pumped. Such pumps are described in U.S. Pat. Nos.5,094,594, 5,730,187, 6,033,628, and 6,533,553, which discloses deviceswhich can pump fluid volumes in the nanoliter or picoliter range.Preferably all materials within the pump that come into contact withsample are made of highly inert materials, e.g., polyetheretherketone(PEEK), fused silica, or sapphire.

A motive force is necessary to move the sample through the capillaryflow cell to push the sample through the interrogation space foranalysis. A motive force is also required to push a flushing samplethrough the capillary flow cell after the sample has been passedthrough. A motive force is also required to push the sample back outinto a sample recovery vessel, when sample recovery is employed.Standard pumps come in a variety of sizes, and the proper size may bechosen to suit the anticipated sample size and flow requirements. Insome embodiments, separate pumps are used for sample analysis and forflushing of the system. The analysis pump may have a capacity ofapproximately 0.000001 mL to approximately 10 mL, or approximately 0.001mL to approximately 1 mL, or approximately 0.01 mL to approximately 0.2mL, or approximately 0.005, 0.01, 0.05, 0.1, or 0.5 mL. Flush pumps maybe of larger capacity than analysis pumps. Flush pumps may have a volumeof about 0.01 mL to about 20 mL, or about 0.1 mL to about 10 mL, orabout 0.1 mL to about 2 mL, or about or about 0.05, 0.1, 0.5, 1, 5, or10 mL. These pump sizes are illustrative only, and those of skill in theart will appreciate that the pump size may be chosen according to theapplication, sample size, viscosity of fluid to be pumped, tubingdimensions, rate of flow, temperature, and other factors well known inthe art. In some embodiments, pumps of the system are driven by steppermotors, which are easy to control very accurately with a microprocessor.

In preferred embodiments, the flush and analysis pumps are used inseries, with special check valves to control the direction of flow. Theplumbing is designed so that when the analysis pump draws up the maximumsample, the sample does not reach the pump itself. This is accomplishedby choosing the ID and length of the tubing between the analysis pumpand the analysis capillary such that the tubing volume is greater thanthe stroke volume of the analysis pump.

4. Detectors

In one embodiment, light (e.g., light in the ultra-violet, visible orinfrared range) emitted by a fluorescent label after exposure toelectromagnetic radiation is detected. The detector 309 (FIG. 1A), ordetectors (364, 365, FIG. 1B), is capable of capturing the amplitude andduration of photon bursts from a fluorescent moiety, and furtherconverting the amplitude and duration of the photon burst to electricalsignals. Detection devices such as CCD cameras, video input modulecameras, and Streak cameras can be used to produce images withcontiguous signals. In another embodiment, devices such as a bolometer,a photodiode, a photodiode array, avalanche photodiodes, andphotomultipliers which produce sequential signals may be used. Anycombination of the aforementioned detectors may also be used. In oneembodiment, avalanche photodiodes are used for detecting photons.

Using specific optics between an interrogation space 314 (FIG. 2A) andits corresponding detector 309 (FIG. 1A), several distinctcharacteristics of the emitted electromagnetic radiation can be detectedincluding: emission wavelength, emission intensity, burst size, burstduration, and fluorescence polarization. In some embodiments, thedetector 309 is a photodiode that is used in reverse bias. A photodiodeset in reverse bias usually has an extremely high resistance. Thisresistance is reduced when light of an appropriate frequency shines onthe P/N junction. Hence, a reverse biased diode can be used as adetector by monitoring the current running through it. Circuits based onthis effect are more sensitive to light than ones based on zero bias.

In one embodiment of the analyzer system, the photodiode can be anavalanche photodiode, which can be operated with much higher reversebias than conventional photodiodes, thus allowing each photo-generatedcarrier to be multiplied by avalanche breakdown, resulting in internalgain within the photodiode, which increases the effective responsiveness(sensitivity) of the device. The choice of photodiode is determined bythe energy or emission wavelength emitted by the fluorescently labeledparticle. In some embodiments, the photodiode is a silicon photodiodethat detects energy in the range of 190-1100 nm; in another embodimentthe photodiode is a germanium photodiode that detects energy in therange of 800-1700 nm; in another embodiment the photodiode is an indiumgallium arsenide photodiode that detects energy in the range of 800-2600nm; and in yet other embodiments, the photodiode is a lead sulfidephotodiode that detects energy in the range of between less than 1000 nmto 3500 nm. In some embodiments, the avalanche photodiode is asingle-photon detector designed to detect energy in the 400 nm to 1100nm wavelength range. Single photon detectors are commercially available(for example Perkin Elmer, Wellesley, Mass.).

In some embodiments the detector is a avalanche photodiode detector thatdetects energy between 300 nm and 1700 nm. In one embodiment, siliconavalanche photodiodes can be used to detect wavelengths between 300 nmand 1100 nm. Indium gallium arsenic photodiodes can be used to detectwavelengths between 900 nm and 1700 nm. In some embodiments, an analyzersystem can comprise at least one detector; in other embodiments, theanalyzer system can comprise at least two detectors, and each detectorcan be chosen and configured to detect light energy at a specificwavelength range. For example, two separate detectors can be used todetect particles that have been tagged with different labels, which uponexcitation with an EM source, will emit photons with energy in differentspectra. In one embodiment, an analyzer system can comprise a firstdetector that can detect fluorescent energy in the range of 450-700 nmsuch as that emitted by a green dye (e.g. Alexa 546); and a seconddetector that can detect fluorescent energy in the range of 620-780 nmsuch as that emitted by a far-red dye (e.g. Alexa 647). Detectors fordetecting fluorescent energy in the range of 400-600 nm such as thatemitted by blue dyes (e.g. Hoechst 33342), and for detecting energy inthe range of 560-700 nm such as that emitted by red dyes (Alexa 546 andCy3) can also be used.

A system comprising two or more detectors can be used to detectindividual particles that are each tagged with two or more labels thatemit light in different spectra. For example, two different detectorscan detect an antibody that has been tagged with two different dyelabels. Alternatively, an analyzer system comprising two detectors canbe used to detect particles of different types, each type being taggedwith a different dye molecules, or with a mixture of two or more dyemolecules. For example, two different detectors can be used to detecttwo different types of antibodies that recognize two different proteins,each type being tagged with a different dye label or with a mixture oftwo or more dye label molecules. By varying the proportion of the two ormore dye label molecules, two or more different particle types can beindividually detected using two detectors. It is understood that threeor more detectors can be used without departing from the scope of theinvention.

It should be understood by one skilled in the art that one or moredetectors can be configured at each interrogation space, whether one ormore interrogation spaces are defined within a flow cell, and that eachdetector may be configured to detect any of the characteristics of theemitted electromagnetic radiation listed above. The use of multipledetectors, e.g., for multiple interrogation spaces, has been previouslydisclosed in a prior application and is incorporated by reference herefrom U.S. patent application Ser. No. 11/048,660. Once a particle islabeled to render it detectable (or if the particle possesses anintrinsic characteristic rendering it detectable), any suitabledetection mechanism known in the art may be used without departing fromthe scope of the present invention, for example a CCD camera, a videoinput module camera, a Streak camera, a bolometer, a photodiode, aphotodiode array, avalanche photodiodes, and photomultipliers producingsequential signals, and combinations thereof. Different characteristicsof the electromagnetic radiation may be detected including: emissionwavelength, emission intensity, burst size, burst duration, fluorescencepolarization, and any combination thereof.

C. Sampling System

In a further embodiment, the analyzer system may include a samplingsystem to prepare the sample for introduction into the analyzer system.The sampling system included is capable of automatically sampling aplurality of samples and providing a fluid communication between asample container and a first interrogation space.

In some embodiments, the analyzer system of the invention includes asampling system for introducing an aliquot of a sample into the singleparticle analyzer for analysis. Any mechanism that can introduce asample may be used. Samples can be drawn up using either a vacuumsuction created by a pump or by pressure applied to the sample thatwould push liquid into the tube, or by any other mechanism that servesto introduce the sample into the sampling tube. Generally, but notnecessarily, the sampling system introduces a sample of known samplevolume into the single particle analyzer; in some embodiments where thepresence or absence of a particle or particles is detected, preciseknowledge of the sample size is not critical. In preferred embodimentsthe sampling system provides automated sampling for a single sample or aplurality of samples. In embodiments where a sample of known volume isintroduced into the system, the sampling system provides a sample foranalysis of more than about 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or 2000 μl.In some embodiments the sampling system provides a sample for analysisof less than about 2000, 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40,30, 20, 10, 5, 2, 1, 0.1, 0.01, or 0.001 μl. In some embodiments thesampling system provides a sample for analysis of between about 0.01 and1500 μl, or about 0.1 and 1000 μl, or about 1 and 500 or about 1 and 100μl, or about 1 and 50 μl, or about 1 and 20 μl. In some embodiments, thesampling system provides a sample for analysis between about 5 μl and200 μl, or about 5 μl and about 100 μl, or about 5 μl and 50 μl. In someembodiments, the sampling system provides a sample for analysis betweenabout 10 μl and 200 μl, or between about 10 μl and 100 μl, or betweenabout 10 μl and 50 μl. In some embodiments, the sampling system providesa sample for analysis between about 0.5 μl and about 50 μl.

In some embodiments, the sampling system provides a sample size that canbe varied from sample to sample. In these embodiments, the sample sizemay be any one of the sample sizes described herein, and may be changedwith every sample, or with sets of samples, as desired.

Sample volume accuracy, and sample to sample volume precision of thesampling system, is required for the analysis at hand. In someembodiments, the precision of the sampling volume is determined by thepumps used, typically represented by a CV of less than about 50, 40, 30,20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01% of sample volume. Insome embodiments, the sample to sample precision of the sampling systemis represented by a CV of less than about 50, 40, 30, 20, 10, 5, 4, 3,2, 1, 0.5, 0.1, 0.05, or 0.01%. In some embodiments, the intra-assayprecision of the sampling system is represented by a CV of less thanabout 10, 5, 1, 0.5, or 0.1%. In some embodiments, the intra-assayprecision of the sampling system shows a CV of less than about 5%. Insome embodiments, the interassay precision of the sampling system isrepresented by a CV of less than about 10, 5, or 1%. In someembodiments, the interassay precision of the sampling system shows a CVof less than about 5%.

In some embodiments, the sampling system provides low sample carryover,advantageous in that an additional wash step is not required betweensamples. Thus, in some embodiments, sample carryover is less than about1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or 0.001%. In someembodiments, sample carryover is less than about 0.02%. In someembodiments, sample carryover is less than about 0.01%.

In some embodiments the sampler provides a sample loop. In theseembodiments, multiple samples are drawn into tubing sequentially andeach is separated from the others by a “plug” of buffer. The samplestypically are read one after the other with no flushing in between.Flushing is done once at the end of the loop. In embodiments where abuffer “plug” is used, the plug may be recovered ejecting the bufferplug into a separate well of a microtiter plate.

The sampling system may be adapted for use with standard assayequipment, for example, a 96-well microtiter plate, or, preferably, a384-well plate. In some embodiments the system includes a 96 well platepositioner and a mechanism to dip the sample tube into and out of thewells, e.g., a mechanism providing movement along the X, Y, and Z axes.In some embodiments, the sampling system provides multiple samplingtubes from which samples may be stored and extracted from, when testingis commenced. In some embodiments, all samples from the multiple tubesare analyzed on one detector. In other embodiments, multiple singlemolecule detectors may be connected to the sample tubes. Samples may beprepared by steps that include operations performed on sample in thewells of the plate prior to sampling by the sampling system, or samplemay be prepared within the analyzer system, or some combination of both.

D. Sample Preparation System

Sample preparation includes the steps necessary to prepare a raw samplefor analysis. These steps can involve, by way of example, one or moresteps of separation steps such as centrifugation, filtration,distillation, chromatography; concentration, cell lysis, alteration ofpH, addition of buffer, addition of diluents, addition of reagents,heating or cooling, addition of label, binding of label, cross-linkingwith illumination, separation of unbound label, inactivation and/orremoval of interfering compounds and any other steps necessary for thesample to be prepared for analysis by the single particle analyzer. Insome embodiments, blood is treated to separate out plasma or serum.Additional labeling, removal of unbound label, and/or dilution steps mayalso be performed on the serum or plasma sample.

In some embodiments, the analyzer system includes a sample preparationsystem that performs some or all of the processes needed to provide asample ready for analysis by the single particle analyzer. This systemmay perform any or all of the steps listed above for sample preparation.In some embodiments samples are partially processed by the samplepreparation system of the analyzer system. Thus, in some embodiments, asample may be partially processed outside the analyzer system first. Forexample, the sample may be centrifuged first. The sample may then bepartially processed inside the analyzer by a sample preparation system.Processing inside the analyzer includes labeling the sample, mixing thesample with a buffer and other processing steps that will be known toone in the art. In some embodiments, a blood sample is processed outsidethe analyzer system to provide a serum or plasma sample, which isintroduced into the analyzer system and further processed by a samplepreparation system to label the particle or particles of interest and,optionally, to remove unbound label. In other embodiments preparation ofthe sample can include immunodepletion of the sample to remove particlesthat are not of interest or to remove particles that can interfere withsample analysis. In yet other embodiments, the sample can be depleted ofparticles that can interfere with the analysis of the sample. Forexample, sample preparation can include the depletion of heterophilicantibodies, which are known to interfere with immunoassays that usenon-human antibodies to directly or indirectly detect a particle ofinterest. Similarly, other proteins that interfere with measurements ofthe particles of interest can be removed from the sample usingantibodies that recognize the interfering proteins.

In some embodiments, the sample can be subjected to solid phaseextraction prior to being assayed and analyzed. For example, a serumsample that is assayed for cAMP can first be subjected to solid phaseextraction using a c18 column to which it binds. Other proteins such asproteases, lipases and phosphatases are washed from the column, and thecAMP is eluted essentially free of proteins that can degrade orinterfere with measurements of cAMP. Solid phase extraction can be usedto remove the basic matrix of a sample, which can diminish thesensitivity of the assay. In yet other embodiments, the particles ofinterest present in a sample may be concentrated by drying orlyophilizing a sample and solubilizing the particles in a smaller volumethan that of the original sample.

In some embodiments the analyzer system provides a sample preparationsystem that provides complete preparation of the sample to be analyzedon the system, such as complete preparation of a blood sample, a salivasample, a urine sample, a cerebrospinal fluid sample, a lymph sample, aBAL sample, a biopsy sample, a forensic sample, a bioterrorism sample,and the like. In some embodiments the analyzer system provides a samplepreparation system that provides some or all of the sample preparation.In some embodiments, the initial sample is a blood sample that isfurther processed by the analyzer system. In some embodiments, thesample is a serum or plasma sample that is further processed by theanalyzer system. The serum or plasma sample may be further processed by,e.g., contacting with a label that binds to a particle or particles ofinterest; the sample may then be used with or without removal of unboundlabel.

In some embodiments, sample preparation is performed, either outside theanalysis system or in the sample preparation component of the analysissystem, on one or more microtiter plates, such as a 96-well plate.Reservoirs of reagents, buffers, and the like can be in intermittentfluid communication with the wells of the plate by means of tubing orother appropriate structures, as are well-known in the art. Samples maybe prepared separately in 96 well plates or tubes. Sample isolation,label binding and, if necessary, label separation steps may be done onone plate. In some embodiments, prepared particles are then releasedfrom the plate and samples are moved into tubes for sampling into thesample analysis system. In some embodiments, all steps of thepreparation of the sample are done on one plate and the analysis systemacquires sample directly from the plate. Although this embodiment isdescribed in terms of a 96-well plate, it will be appreciated that anyvessel for containing one or more samples and suitable for preparationof sample may be used. For example, standard microtiter plates of 384 or1536 wells may be used. More generally, in some embodiments, the samplepreparation system is capable of holding and preparing more than about5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 1000, 5000,or 10,000 samples. In some embodiments, multiple samples may be sampledfor analysis in multiple analyzer systems. Thus, in some embodiments, 2samples, or more than about 2, 3, 4, 5, 7, 10, 15 20, 50, or 100 samplesare sampled from the sample preparation system and run in parallel onmultiple sample analyzer systems.

Microfluidics systems may also be used for sample preparation and assample preparation systems that are part of analyzer systems, especiallyfor samples suspected of containing concentrations of particles highenough that detection requires smaller samples. Principles andtechniques of microfluidic manipulation are known in the art. See, e.g.,U.S. Pat. Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681,751;5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410;5,543,838; 5,480,614, 5,716,825; 5,603,351; 5,858,195; 5,863,801;5,955,028; 5,989,402; 6,041,515; 6,071,478; 6,355,420; 6,495,104;6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744;6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992;6,537,437; 6,599,436; 6,811,668 and published PCT patent application no.WO9955461(A1). Samples may be prepared in series or in parallel, for usein a single or multiple analyzer systems.

Preferably, the sample comprises a buffer. The buffer may be mixed withthe sample outside the analyzer system, or it may be provided by thesample preparation mechanism. While any suitable buffer can be used, thepreferable buffer has low fluorescence background, is inert to thedetectably labeled particle, can maintain the working pH and, inembodiments wherein the motive force is electrokinetic, has suitableionic strength for electrophoresis. The buffer concentration can be anysuitable concentration, such as in the range from about 1 to about 200mM. Any buffer system may be used as long as it provides for solubility,function, and delectability of the molecules of interest. Preferably,for application using pumping, the buffer is selected from the groupconsisting of phosphate, glycine, acetate, citrate, acidulate,carbonate/bicarbonate, imidazole, triethanolamine, glycine amide,borate, MES, Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane, BES,MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS,Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO,AMP, CAPS, and CABS. The buffer can also be selected from the groupconsisting of Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid(MES), 4-morpholine propanesulfonic acid (MOPS) and2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is 2 mMTris/borate at pH 8.1, but Tris/glycine and Tris/HCl are alsoacceptable. Other buffers are as described herein.

Buffers useful for electrophoresis are disclosed in a prior applicationand are incorporated by reference herein from U.S. patent applicationSer. No. 11/048,660.

E. Sample Recovery

One highly useful feature of embodiments of the analyzers and analysissystems of the invention is that the sample can be analyzed withoutconsuming it. This can be especially important when sample materials arelimited. Recovering the sample also allows one to do other analyses orreanalyze it. The advantages of this feature for applications wheresample size is limited and/or where the ability to reanalyze the sampleis desirable, e.g., forensic, drug screening, and clinical diagnosticapplications, will be apparent to those of skill in the art.

Thus, in some embodiments, the analyzer system of the invention furtherprovides a sample recovery system for sample recovery after analysis. Inthese embodiments, the system includes mechanisms and methods by whichthe sample is drawn into the analyzer, analyzed and then returned, e.g.,by the same path, to the sample holder, e.g., the sample tube. Becauseno sample is destroyed and because it does not enter any of the valvesor other tubing, it remains uncontaminated. In addition, because all thematerials in the sample path are highly inert, e.g., PEEK, fused silica,or sapphire, there is little contamination from the sample path. The useof the stepper motor controlled pumps (particularly the analysis pump)allows precise control of the volumes drawn up and pushed back out. Thisallows complete or nearly complete recovery of the sample with little ifany dilution by the flush buffer. Thus, in some embodiments, more thanabout 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% of the sample is recovered after analysis. In some embodiments,the recovered sample is undiluted. In some embodiments, the recoveredsample is diluted less than about 1.5-fold, 1.4-fold, 1.3-fold,1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1.005-fold, or 1.001-fold.

For sampling and/or sample recovery, any mechanism for transporting aliquid sample from a sample vessel to the analyzer may be used. In someembodiments the inlet end of the analysis capillary has attached a shortlength of tubing, e.g., PEEK tubing that can be dipped into a samplecontainer, e.g. a test tube or sample well, or can be held above a wastecontainer. When flushing, to clean the previous sample from theapparatus, this tube is positioned above the waste container to catchthe flush waste. When drawing a sample in, the tube is put into thesample well or test tube. Typically the sample is drawn in quickly, andthen pushed out slowly while observing particles within the sample.Alternatively, in some embodiments, the sample is drawn in slowly duringat least part of the draw-in cycle; the sample may be analyzed whilebeing slowly drawn in. This can be followed by a quick return of thesample and a quick flush. In some embodiments, the sample may beanalyzed both on the inward (draw-in) and outward (pull out) cycle,which improves counting statistics, e.g., of small and dilute samples,as well as confirming results, and the like. If it is desired to savethe sample, it can be pushed back out into the same sample well it camefrom, or to another. If saving the sample is not desired, the tubing ispositioned over the waste container.

VI. Methods Using Highly Sensitive Analysis of Molecules

The systems, system kits, and methods of the present invention makepossible measurement of molecules in samples at concentrations far lowerthan previously measured. The high sensitivity of the instruments, kits,and methods of the invention allows the establishment of markers, e.g.,biological markers, that have not previously been possible because of alack of sensitivity of detection. The invention also includes the use ofthe compositions and methods described herein for the discovery of newmarkers.

There are numerous markers currently available which, while potentiallyof use in determining a biological state, are not currently of practicaluse because their lower ranges are unknown. In some cases, abnormallyhigh levels of the marker are detectable by current methodologies, butnormal ranges have not been established. In some cases, upper normalranges of the marker are detectable, but not lower normal ranges, orlevels below normal. In some cases, for example, markers specific totumors, or markers of infection, any level of the marker indicates thepotential presence of the biological state, and enhancing sensitivity ofdetection is an advantage for early diagnosis. In some cases, the rateof change, or lack of change, in the concentration of the marker overmultiple timepoints provides the most useful information, but presentmethods of analysis do not permit determination of levels of the markerat timepoint sampling in the early stages of a condition, when it istypically at its most treatable. In many cases, the marker may bedetected at clinically useful levels only through the use of cumbersomemethods that are not practical or useful in a clinical setting, such asmethods that require complex sample treatment and time-consuminganalysis.

In addition, there are potential markers of biological states that existin sufficiently low concentrations that their presence remains extremelydifficult or impossible to detect by current methods.

The analytical methods and compositions of the present invention providelevels of sensitivity and precision that allow the detection of markersfor biological states at concentrations at which the markers have beenpreviously undetectable, thus allowing the “repurposing” of such markersfrom confirmatory markers, or markers useful only in limited researchsettings, to diagnostic, prognostic, treatment-directing, or other typesof markers useful in clinical settings and/or in large-scale clinicalsettings such as clinical trials. Such methods allow, e.g., thedetermination of normal and abnormal ranges for such markers.

The markers thus repurposed can be used for, e.g., detection of normalstate (normal ranges), detection of responder/non-responder (e.g., to atreatment, such as administration of a drug); early disease orpathological occurrence detection (e.g., detection of cancer in itsearliest stages, early detection of cardiac ischemia); disease staging(e.g., cancer); disease monitoring (e.g., diabetes monitoring,monitoring for recurrence of cancer after treatment); study of diseasemechanism; and study of treatment toxicity, such as toxicity of drugtreatments (e.g., cardiotoxicity).

A. Methods

The invention thus provides methods and compositions for the sensitivedetection of markers, and further methods of establishing values fornormal and abnormal levels of the markers. In further embodiments, theinvention provides methods of diagnosis, prognosis, and/or treatmentselection based on values established for the markers. The inventionalso provides compositions for use in such methods, e.g., detectionreagents for the ultrasensitive detection of markers.

In some embodiments, the invention provides a method of establishing amarker for a biological state, by establishing a range of concentrationsfor the marker in biological samples obtained from a first population bymeasuring the concentrations of the marker the biological samples bydetecting single molecules of the marker (e.g., by detecting a labelthat has been attached to a single molecule of the marker). In someembodiments, the marker is a polypeptide or small molecule. The samplesmay be any sample type described herein, e.g., blood, plasma, or serum;or urine.

The method may utilize samples from a first population where thepopulation is a population that does not exhibit the biological state.In the case where the biological state is a disease state, the firstpopulation may be a population that does not exhibit the disease, e.g.,a “normal” population. In some embodiments the method may furthercomprise establishing a range of range of levels for the marker inbiological samples obtained from a second population, where the membersof the second population exhibit the biological state, by measuring theconcentrations of the marker the biological samples by detecting singlemolecules of the marker. In some embodiments, e.g., cross-sectionalstudies, the first and second populations are different. In someembodiments, at least one member of the second population is a member ofthe first population, or at least one member of said the population is amember of the second population. In some embodiments, e.g., longitudinalstudies, substantially all the members of the second population aremembers of the first population who have developed the biological state(e.g., a disease or pathological state).

The detecting of single molecules of the marker is performed using amethod as described herein, e.g., a method with a limit of detection forsaid marker of less than about 1000, 100, 50, 20, 10, 5, 1, 0.5, 0.1,0.05, 0.01, 0.005, or 0.001 femtomolar of the marker in the samples, bydetecting single molecules of the marker.

The biological state may be a phenotypic state; a condition affectingthe organism; a state of development; age; health; pathology; disease;disease process; disease staging; infection; toxicity; or response tochemical, environmental, or drug factors (such as drug responsephenotyping, drug toxicity phenotyping, or drug effectivenessphenotyping).

In some embodiments, the biological state is a pathological state,including but not limited to inflammation, abnormal cell growth, andabnormal metabolic state. In some embodiments, the state is a diseasestate. Disease states include, but are not limited to, cancer,cardiovascular disease, inflammatory disease, autoimmune disease,neurological disease, infectious disease and pregnancy relateddisorders. In some embodiments the state is a disease stage state, e.g.,a cancer disease stage state.

The methods may also be used for determination of a treatment responsestate. In some embodiments, the treatment is a drug treatment. Theresponse may be a therapeutic effect or a side effect, e.g., an adverseeffect. Markers for therapeutic effects will be based on the disease orcondition treated by the drug. Markers for adverse effects typicallywill be based on the drug class and specific structure and mechanism ofaction and metabolism. A common adverse effect is drug toxicity. Anexample is cardiotoxicity, which can be monitored by the marker cardiactroponin. In some embodiments one or more markers for the disease stateand one or more markers for one or more adverse effects of a drug aremonitored, typically in a population that is receiving the drug. Samplesmay be taken at intervals and the respective values of the markers inthe samples may be evaluated over time.

The detecting of single molecules of the marker may comprise labelingthe marker with a label comprising a fluorescent moiety capable ofemitting at least about 200 photons when simulated by a laser emittinglight at the excitation wavelength of the moiety, where the laser isfocused on a spot not less than about 5 microns in diameter thatcontains the moiety, and wherein the total energy directed at the spotby the laser is no more than about 3 microJoules. In some embodiments,the fluorescent moiety comprises a molecule that comprises at least onesubstituted indolium ring system in which the substituent on the3-carbon of the indolium ring contains a chemically reactive group or aconjugated substance. In some embodiments, the fluorescent moiety maycomprise a dye selected from the group consisting of AlexaFluor 488,AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. Insome embodiments, the moiety comprises AlexaFluor 647. In someembodiments, the label further comprises a binding partner for themarker, e.g., an antibody specific for said marker, such as a polyclonalantibody or a monoclonal antibody. Binding partners for a variety ofmarkers are described herein.

The method may further include establishing a threshold level for themarker based on the first range, or the first and second ranges, wherethe presence of marker in a biological sample from an individual at alevel above or below the threshold level indicates an increasedprobability of the presence of the biological state in said individual.An example of a threshold determined for a normal population is thesuggested threshold for cardiac troponin of greater than the 99thpercentile value in a normal population. See Example 3. Other thresholdlevels may be determined empirically, i.e., based on data from the firstand second populations regarding marker levels and the presence,absence, severity, rate of progression, rate of regression, and thelike, of the biological state being monitored. It will be appreciatedthat threshold levels may be established at either end of a range, e.g.,a minimum below which the concentration of the marker in a sampleindicates an increased probability of a biological state, and/or amaximum above which the concentration of the marker in a sampleindicates an increased probability of a biological state. In someembodiments, a risk stratification may be produced in which two or moreranges of marker concentrations correspond to two or more levels ofrisk. Other methods of analyzing data from two populations and formarkers and producing clinically-relevant values for use by, e.g.,physicians and other health care professionals, are well-known in theart.

For some biological markers, the presence of any marker at all is anindication of a disease or pathological state, and the threshold isessentially zero. An example is the use of prostate specific antigen(PSA) to monitor cancer recurrence after removal of the prostate gland.As PSA is produced only by the prostate gland, and as the prostate glandand all tumor are presumed to be removed, PSA after removal is zero.Appearance of PSA at any level signals a possible recurrence of thecancer, e.g., at a metastatic site. Thus, the more sensitive the methodof detection, the earlier an intervention may be made should suchrecurrence occur.

Other evaluations of marker concentration may also be made, such as in aseries of samples, where change in value, rate of change, spikes,decrease, and the like may all provide useful information fordetermination of a biological state. In addition, panels of markers maybe used if it is found that more than one marker provides informationregarding a biological state. If panels of markers are used, the markersmay be measured separately in separate samples (e.g., aliquots of acommon sample) or simultaneously by multiplexing. Examples of panels ofmarkers and multiplexing are given in, e.g., U.S. patent applicationSer. No. 11/048,660.

The establishment of such markers and, e.g., reference ranges for normaland/or abnormal states, allow for sensitive and precise determination ofthe biological state of an organism. Thus, in some embodiments, theinvention provides a method for detecting the presence or absence of abiological state of an organism, comprising i) measuring theconcentration of a marker in a biological sample from the organism,wherein said marker is a marker established through establishing a rangeof concentrations for said marker in biological samples obtained from afirst population by measuring the concentrations of the marker thebiological samples by detecting single molecules of the marker; and ii)determining the presence of absence of said biological state based onsaid concentration of said marker in said organism.

In some embodiments, the invention provides a method for detecting thepresence or absence of a biological state in an organism, comprising i)measuring the concentrations of a marker in a plurality of biologicalsamples from said organism, wherein said marker is a marker establishedthrough establishing a range of concentrations for said marker inbiological samples obtained from a first population by measuring theconcentrations of the marker the biological samples by detecting singlemolecules of the marker; and ii) determining the presence of absence ofsaid biological state based on said concentrations of said marker insaid plurality of samples. In some embodiments, the samples are ofdifferent types, e.g., are samples from different tissue types. In thiscase, the determining is based on a comparison of the concentrations ofsaid marker in said different types of samples. More commonly, thesamples are of the same type, and the samples are taken at intervals.The samples may be any sample type described herein, e.g., blood,plasma, or serum; or urine. Intervals between samples may be minutes,hours, days, weeks, months, or years. In an acute clinical setting, theintervals may be minutes or hours. In settings involving the monitoringof an individual, the intervals may be days, weeks, months, or years.

In many cases, the biological state whose presence or absence is to bedetected is a disease phenotype. Thus, in one embodiment, a phenotypicstate of interest is a clinically diagnosed disease state. Such diseasestates include, for example, cancer, cardiovascular disease,inflammatory disease, autoimmune disease, neurological disease,respiratory disease, infectious disease and pregnancy related disorders.

Cancer phenotypes are included in some aspects of the invention.Examples of cancer herein include, but are not limited to: breastcancer, skin cancer, bone cancer, prostate cancer, liver cancer, lungcancer, brain cancer, cancer of the larynx, gallbladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, non-small cell lungcarcinoma gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Cardiovascular disease may be included in other applications of theinvention. Examples of cardiovascular disease include, but are notlimited to, congestive heart failure, high blood pressure, arrhythmias,atherosclerosis, cholesterol, Wolff-Parkinson-White Syndrome, long QTsyndrome, angina pectoris, tachycardia, bradycardia, atrialfibrillation, ventricular fibrillation, congestive heart failure,myocardial ischemia, myocardial infarction, cardiac tamponade,myocarditis, pericarditis, arrhythmogenic right ventricular dysplasia,hypertrophic cardiomyopathy, Williams syndrome, heart valve diseases,endocarditis, bacterial, pulmonary atresia, aortic valve stenosis,Raynaud's disease, Raynaud's disease, cholesterol embolism, Wallenbergsyndrome, Hippel-Lindau disease, and telangiectasis.

Inflammatory disease and autoimmune disease may be included in otherembodiments of the invention. Examples of inflammatory disease andautoimmune disease include, but are not limited to, rheumatoidarthritis, non-specific arthritis, inflammatory disease of the larynx,inflammatory bowel disorder, psoriasis, hypothyroidism (e.g., Hashimotothyroidism), colitis, Type 1 diabetes, pelvic inflammatory disease,inflammatory disease of the central nervous system, temporal arteritis,polymyalgia rheumatica, ankylosing spondylitis, polyarteritis nodosa,Reiter's syndrome, scleroderma, systemic lupus and erythematosus.

The methods and compositions of the invention can also providelaboratory information about markers of infectious disease includingmarkers of Adenovirus, Bordetella pertussis, Chlamydia pneumoniae,Chlamydia trachomatis, Cholera Toxin, Cholera Toxin β, Campylobacterjejuni, Cytomegalovirus, Diptheria Toxin, Epstein-Barr NA, Epstein-BarrEA, Epstein-Barr VCA, Helicobacter Pylori, Hepatitis B virus (HBV) Core,Hepatitis B virus (HBV) Envelope, Hepatitis B virus (HBV) Survace (Ay),Hepatitis C virus (HCV) Core, Hepatitis C virus (HCV) NS3, Hepatitis Cvirus (HCV) NS4, Hepatitis C virus (HCV) NS5, Hepititis A, Hepititis D,Hepatitis E virus (HEV) orf2 3 KD, Hepatitis E virus (HEV) orf2 6 KD,Hepatitis E virus (HEV) orf3 3 KD, Human immunodeficiency virus (HIV)-1p24, Human immunodeficiency virus (HIV)-1 gp41, Human immunodeficiencyvirus (HIV)-1 gp120, Human papilloma virus (HPV), Herpes simplex virusHSV-1/2, Herpes simplex virus HSV-1 gD, Herpes simplex virus HSV-2 gG,Human T-cell leukemia virus (HTLV)-1/2, Influenza A, Influenza A H3N2,Influenza B, Leishmania donovani, Lyme disease, Mumps, M. pneumoniae, M.tuberculosis, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, PolioVirus, Respiratory syncytial virus (RSV), Rubella, Rubeola, StreptolysinO, Tetanus Toxin, T. pallidum 15 kd, T. pallidum p47, T. cruzi,Toxoplasma, and Varicella Zoster.

B. Exemplary Markers

The instruments, labels, and methods of the invention have been used toestablish ranges for markers in, e.g., serum and urine, at levels 10- to100-fold lower than previous levels, or lower. The markers areindicative of a wide variety of biological states, e.g., cardiac diseaseand cardiotoxicity (troponin), infection (TREM-1), inflammation andother conditions (LTE4, IL-6 and IL-8), asthma (LTE4), cancer (Akt1,TGF-beta, Fas ligand), and allograft rejection and degenerative disease(Fas ligand).

Markers include protein and non-protein markers. The markers aredescribed briefly here and procedures and results given in the Examples.

1. Cardiac Damage

Cardiac troponin is an example of a marker that is currently detectableonly in abnormally high amounts. Cardiac troponin is a marker of cardiacdamage, useful in diagnosis, prognosis, and determination of method oftreatment in a number of diseases and conditions, e.g., acute myocardialinfarct. In addition, cardiac troponin is a useful marker ofcardiotoxicity due to treatment, e.g., drug treatment.

The troponin complex in muscle consists of troponin I, C and T. TroponinC exists as two isoforms, one from cardiac and slow-twitch muscle andone from fast-twitch muscle; because it is found in virtually allstriated muscle, its use as a specific marker is limited. In contrast,troponin I and T are expressed as different isoforms in slow-twitch,fast-twitch and cardiac muscle The unique cardiac isoforms of troponin Iand T allow them to be distinguished immunologically from the othertroponins of skeletal muscle. Therefore, the release into the blood ofcardiac troponin I and T is indicative of damage to cardiac muscle, andprovides the basis for their use as diagnostic or prognostic markers, orto aid in determination of treatment.

Currently used markers for cardiac damage suffer disadvantages thatlimit their clinical usefulness. Cardiac enzyme assays have formed thebasis for determining whether or not there is damage to the cardiacmuscle. Unfortunately, the standard creatine kinase-MB (CK-MB) assay isnot reliable in excluding infarction until 10 to 12 hours after theonset of chest pain. Earlier diagnosis would have very specificadvantages with regard to fibrinolytic therapy and triage.

Because the level of troponin found in the circulation of healthyindividuals is very low, and cardiac specific troponins do not arisefrom extra-cardiac sources, the troponins are very sensitive andspecific markers of cardiac injury. In addition to cardiac infarct, anumber of other conditions can cause damage to the heart muscle, andearly detection of such damage would prove useful to clinicians.However, present methods of detection and quantitation of cardiactroponin do not possess sufficient sensitivity to detect the release ofcardiac troponin into the blood until levels have reached abnormallyhigh concentrations, e.g., 0.1 ng/ml or greater.

The methods and compositions of the invention thus include methods andcompositions for the highly sensitive detection and quantitation ofcardiac troponin, and compositions and methods for diagnosis, prognosis,and/or determination of treatment based on such highly sensitivedetection and quantitation. A standard curve for cardiac troponin I wasestablished with a limit of detection less than about 1 pg/l (Example1). Levels of cardiac troponin I were established in normal individuals,and a threshold value at the 99^(th) percentile of normal established(Example 3). Serial samples from individuals who suffered acutemyocardial infarct were analyzed, and time courses for cardiac troponinI concentrations, including deviations from baseline, were determined(Example 4). Thus, cardiac troponin I serves as an example of a markerthat can be detected by the systems and methods of the invention atlevels to provide diagnostic and prognostic information of use inclinical and research settings. See also U.S. patent application Ser.No. _(——————), entitled “Highly Sensitive System and Methods forAnalysis of Troponin,” filed on even date herewith, which isincorporated by reference herein in its entirety.

2. Infection

TREM-1 is a marker of bacterial or fungal infections. Assays of theinvention suggest that TREM-1 may routinely be measured at aconcentration of 100 fM

3. Cytokines

The normal level of many cytokines, chemokines and growth factors is notknown primarily because of the inability of existing technology todetect levels that are below those found in samples from diseasedpatients. For example, the basal level of other cytokines such as IL-10,TNF-alpha, IL-4, IL-1beta, IL-2, IL-12 and IFN-gamma cannot be detectedby routine assays that are performed in a clinical setting, whereas theanalyzer systems of the invention can readily determine the level ofthese and other cytokines. Knowing the level of cytokines and growthfactors aids clinicians with the diagnosis, prognosis and treatment of avariety of diseases including cancer, and respiratory, infectious, andcardiovascular diseases. Early cytokine detection to monitor normal anddisease states in clinical specimens can be achieved using the analyzersystems of the invention to analyze samples such as plasma, serum, andurine as well as other fluid samples to provide for better translationalmedicine. For example, determining levels of cytokines for which anormal range of concentration is not known, would aid clinicians withdiagnosis and treatment of the following conditions and diseases. BoneMorphogenetic Proteins would be useful to monitor the treatment forfractures, spinal fusions, orthopedic surgery, and oral surgery;Interleukin-10 (IL-10) would be useful for detecting and monitoring forthe presence of cancers including non-Hodgkin's lymphoma, multiplemyeloma, melanoma, and ovarian cancer, as well as for detecting andmonitoring the effect of anti-inflammatory therapy, organtransplantation, immunodeficiencies, and parasitic infections;Interleukin-11 (IL-11) is useful for the detection and monitoring forthe presence of cancers such as breast cancer; Interleukin-12 (IL-12)for cancer and HIV infections; TNFα, an inflammatory cytokine, alone orin combination with IL-6, can be used as a good predictor of sepsis,acute pancreatitis, tuberculosis, and autoimmune disease such asrheumatoid arthritis and lupus.

Alternatively, databases may already exist for normal and abnormalvalues but present methods may not be practical for screeningindividuals on a routine basis to determine with sufficient sensitivitywhether the value of the individual for the marker is within the normalrange. For example, most present methods for the determination of IL-6concentration in a sample are capable of detecting IL-6 only down to aconcentration of about 5 pg/ml; the normal range of IL-6 values is about1 to about 10 pg/ml; hence, present methods are able to detect IL-6 onlyin the upper part of normal ranges. In contrast, the analyzers andanalyzer systems of the invention allow the detection of IL-6 down to aconcentration below about 0.1 pg/ml, or less than one-tenth of normalrange values. The lower limit of quantitation (LLOQ) is about 0.01pg/ml. Thus, the analyzers and analyzer systems of the invention allow afar broader and more nuanced database to be produced for a biomarker,e.g., for IL-6, and also allow screening for that biomarker both withinand outside of the normal range, allowing earlier detection.

Thus, the analyzers and analyzer systems of the invention allow a farbroader and more nuanced database to be produced for a biomarker, e.g.,for IL-6, and also allow screening for that biomarker both within andoutside of the normal range, allowing earlier detection of conditions inwhich IL-6 is implicated. IL-6-related disorders include but are notlimited to sepsis, peripheral arterial disease, and chronic obstructivepulmonary disease. Interleukin-6 mediated inflammation is also thecommon causative factor and therapeutic target for atheroscleroticvascular disease and age-related disorders including osteoporosis andtype 2 diabetes. In addition, IL-6 can be measured in combination withother cytokines, for example TNFα to diagnose additional diseases suchas septic shock.

4. Inflammatory Markers

Other cytokines that can be useful in detecting early onset ofinflammatory disease include markers and panels of markers ofinflammation as described herein. Examples of cytokines that can be usedto detect an inflammatory disorders are Leukotriene 4 (LTE4), which canbe an early marker of asthma and TGFβ, which can be used to detect andmonitor the status of inflammatory disorders including fibrosis,sclerosis. Some markers can be used to detect more than one disorder,for example TGFβ can also be used to detect the presence of cancer.

Leukotriene E4 Cysteinyl leukotrienes (LTC₄, LTD₄, LTE₄) play animportant role in the pathogenesis of asthma. Leukotrienes are producedby mast cells, eosinophils, and other airway inflammatory cells andincrease vascular permeability, constrict bronchial smooth muscle, andmediate bronchial hyperresponsiveness. Levels of urinary LTE₄, thestable metabolite of LTC₄ and LTD₄, are increased in children and adultswith asthma compared with healthy controls and in asthmatics afterbronchial challenge with antigen, after oral challenge with aspirin inaspirin sensitive asthmatic subjects, and during exercise inducedbronchospasm. The importance of leukotrienes in the pathology of asthmahas been further demonstrated in large clinical trials with agents thatblock the actions of leukotrienes. For example, montelukast, a potentleukotriene receptor antagonist taken orally once daily, significantlyimproves asthma control in both children (aged 2-14 years) and adultsand attenuates exercise induced bronchoconstriction.

Activation of the leukotriene pathways is accompanied by rises inurinary levels of LTE4, and acute exacerbations of asthma areaccompanied by increased levels of LTE4 in urine followed by asignificant decrease during resolution. The degree of airflow limitationcorrelates with levels of urinary LTE4 during the exacerbation andfollow up periods, thus indicating that the leukotriene pathway isactivated during acute asthma. In addition, inhalation ofbronchoconstricting doses of LTC4 or LTE4 alter urinary LTE4 excretionin a dose-dependent manner thus indicating that urinary LTE4 can be usedas a marker of sulphidopeptide leukotriene synthesis in the lungs ofpatients with asthma.

The methods of the invention can be used to detect changes in LTE4 inbiological samples such as urinary samples (Examples). Measurements ofsubnanogram levels of LTE4 can be useful as a references for detectingand monitoring sulphidopeptide leukotriene synthesis in the lungs ofpatients with chronic or acute asthma.

6. TGFβ

The methods of the invention can also be performed to detect the earlyonset of diseases for which TGFβ is a marker. Examples of TGFβ-relateddiseases include fibrotic diseases. Fibrosis refers to the excessive andpersistent formation of scar tissue, which is responsible for morbidityand mortality associated with organ failure in a variety of chronicdiseases affecting the lungs, kidneys, eyes, heart, liver, and skin.TGFβ is well known for its role as a mediator of chronic fibroticeffects. For example, TGFβ is implicated in promoting fibroblasticproliferation and matrix accumulation in fibrotic lung disease.Inhibition of TGFβ has been proposed as a potential therapeutic avenuefor the management of lung fibrosis. TGFβ not only stimulates thesynthesis of many extracellular matrix molecules, including fibronectinand type I collagen and their receptors, but also decreases matrixdegradation via differential effects on the expression of proteases andtheir inhibitors, strongly promoting generation of extracellular matrix.Thus the analyzer systems of the invention can detect abnormal levels ofTGFβ, e.g., associated with fibrotic diseases, including but not limitedto idiopathic pulmonary fibrosis, diabetic nephropathy, progressivenephropathies including glomerulosclerosis and IgA nephropathy (causesof kidney failure and the need for dialysis and retransplant); diabeticretinopathy and advanced macular degeneration (fibrotic diseases of theeye and leading causes of blindness); cirrhosis and biliary atresia(leading causes of liver fibrosis and failure); and congestive heartfailure, myocardiopathy associated with progressive fibrosis in Chagasdisease; lung fibrosis; and scleroderma.

TGFβ is also a marker for cancers including prostate cancer, cervicalcancer, lung carcinoma, and Hodgkin's disease. Plasma levels of TGFβ inpatients with lung cancer are often elevated. It has been shown that inpatients with an elevated plasma TGF beta 1 level at diagnosis,monitoring this level may be useful in detecting both diseasepersistence and recurrence after radiotherapy.

Transforming growth factor-beta (TGF-beta) is a multipotent growthfactor affecting development, homeostasis, and tissue repair. Inaddition, increased expression of TGF-beta has been reported indifferent malignancies, suggesting a role for this growth factor intumorigenesis. In particular, it has been demonstrated that the presenceof TGF-beta in the endothelial and perivascular layers of small vesselsin the tumor stroma of osteosarcomas suggests an angiogenic activity ofthis growth factor, and that increased expression of TGF-beta isoformshave been suggested to play a role in the progression of osteosarcoma(Kloen et al., Cancer, 80:2230-9 (1997)). TGFβ is one of the few knownproteins that can inhibit cell growth, however, although the notion iscontroversial, some researchers believe that some human malignanciessuch as breast cancer subvert TGFβ for their own purposes. In a paradoxthat is not understood, these cancers make TGFβ and steadily increaseits expression until it becomes a marker of advancing metastasis anddecreased survival. For example, levels of plasma TGFβ are markedlyelevated in men with prostate cancer metastatic to regional lymph nodesand bone. In men without clinical or pathologic evidence of metastases,the preoperative plasma TGF-β level is a strong predictor of biochemicalprogression after surgery, presumably because of an association withoccult metastatic disease present at the time of radical prostatectomy.

Other markers of abnormal cell growth that are detected by the methodsof the invention include Akt1, Fas ligand and IL-6.

Diagnosis of cancers often depends on the use of crude measurements oftumor growth, such as visualization of the tumor itself, that are eitherinaccurate or that must reach high levels before they become detectable,e.g., in a practical clinical setting by present methods. At the pointof detection, the tumor has often grown to sufficient size thatintervention is unlikely to occur before metastasis. For example,detection of lung cancer by X-ray requires a tumor of >1 cm in diameter,and by CT scan of >2-3 mm. Alternatively, a biomarker of tumor growthmay be used, but, again, often the tumor is well-advanced by the timethe biomarker is detectable at levels accessible to current clinicaltechnology. Furthermore, after intervention (e.g., surgery,chemotherapy, or radiation to shrink or remove the tumor or tumors), itis often not possible to measure the tumor marker with sufficientsensitivity to determine if there has been a recurrence of the canceruntil residual disease has progressed to the point where furtherintervention is unlikely to be successful. Using the analyzers, systems,and methods of the present invention, it is possible to both detectonset of tumor growth and return of tumor growth at a point whereintervention is more likely to be successful, e.g., due to lowerprobability of metastasis. Markers for cancer that can be detected atlevels not previously shown include markers disclosed above. Examples ofassays for the detection of markers that can be repurposed to diagnosticmarkers include TGFβ discussed above, Akt1, Fas ligand and IL-6, whichare given in Examples.

7. Akt1

Akt1 is v-akt murine thymoma viral oncogene homolog 1 and is aserine-threonine protein kinase encoded by the AKT1gene. Akt kinaseshave been implicated in disparate cell responses, including inhibitionof apoptosis and promotion of cell proliferation, angiogenesis, andtumor cell invasiveness.

Best known for its ability to inhibit apoptotic and non-apoptotic celldeath, Akt can be monitored to predict tumor response to anticancertreatment. Predicting tumor response by assessing the influence ofapoptosis and nonapoptotic cell death, would allow for developing a moreefficient strategy for enhancing the therapeutic effect of anticancertreatment. Anticancer treatment-induced apoptosis is regulated by thebalance of proapoptotic and antiapoptotic proteins through mitochondria,and resistance to apoptosis is mediated by Akt-dependent andBcl-2-dependent pathways. Bcl-2 partially inhibits nonapoptotic celldeath as well as apoptosis, whereas Akt inhibits both apoptotic andnonapoptotic cell death through several target proteins. Since drugsensitivity is likely correlated with the accumulation of apoptotic andnonapoptotic cell death's, which may influence overall tumor response inanticancer treatment. the ability to predict overall tumor response fromthe modulation of several important cell death-related proteins mayresult in a more efficient strategy for improving the therapeuticeffect.

Akt1 is also involved in Epithelial-mesenchymal transition (EMT), whichis an important process during development and oncogenesis by whichepithelial cells acquire fibroblast-like properties and show reducedintercellular adhesion and increased motility. AKT is activated in manyhuman carcinomas, and the AKT-driven EMT may confer the motilityrequired for tissue invasion and metastasis. Thus future therapies basedon AKT inhibition may complement conventional treatments by controllingtumor cell invasion and metastasis. Akt is constitutively activated inmost melanoma cell lines and tumor samples of different progressionstages, and activation of AKT has been linked to the expression ofinvasion/metastasis-related melanoma cell adhesion molecule (MelCAM),which in turn is strongly associated with the acquisition of malignancyby human melanoma. Akt1 is also activated in pancreatic cancer, and AKTactivation has been shown to correlate with higher histologic tumorgrade. Thus, AKT activation is associated with tumor grade, an importantprognostic factor. Akt1 is also upregulated in prostate cancer and thatexpression is correlated with tumor progression. Thus, Akt1 could betargeted for therapeutic intervention of cancer while at its earlieststages. In some embodiments, the analyzer systems of the inventionprovide a method for providing an early diagnosis of a cancer bydetermining the presence or concentration of Akt1 in a sample from apatient when the level of Akt1 is less than about 100, 50, or 25 pg/ml.See Example 6.

8. Fas Ligand

Fas Ligand (FasL), also known as CD95L, is a member of the TNF familyand induces apoptosis via binding to Fas (CD95). The protein exists intwo forms; either membrane FasL or soluble FasL, which migrate atmolecular weight of 45 kDa and 26 kDa, respectively. FasL is expressedon a variety of cells including activated lymphocytes, natural killercells and monocytes. Interaction of FasL and Fas plays an important rolein physiological apoptotic processes. Malfunction of the Fas-FasL systemcauses hyperplasia in peripheral lymphoid organs and acceleratesautoimmune disease progression and tumorigenesis. There are limited dataabout the levels of soluble apoptotic factors in general, and morespecifically about their modulation with therapeutic regimens.

The systems and methods of the invention can detect concentrations ofFas ligand that are as low as 2.4 pg/ml. Thus, in some embodiments, theanalyzer systems and methods of the invention provide for the detectionof Fas ligand to identify pathological conditions such as abnormallevels of apoptosis. Measurements of Fas in patient samples can be usedto diagnose conditions such as polycystic ovarian syndrome, tumors suchas testicular germ cell tumors, bladder cancer, lung cancer, and raretumors such as follicular dendritic cell tumors. In addition, Fasmeasurements of Fas ligand can be used t diagnose allograft rejection;and degenerative disease such as osteoarthritis. Thus, in someembodiments, the analyzer systems and methods of the invention can beused to determine the concentration of Fas ligand in a sample from apatient suspected of suffering from Fas ligand related disorder todiagnose the disorder, or the concentration of Fas ligand can be used tomonitor the progress or status of a Fas ligand related disorder in apatient undergoing therapy for the disorder. In some embodiments, theassay is capable of determining the level of Fas ligand in the sample ata concentration less than about 100, 50, 25, 10, or 5 pg/ml. See Example8.

C. Business Methods

The present invention relates to systems and methods (including businessmethods) for establishing markers that can be used for diagnosing abiological state or a condition in an organism, preparing diagnosticsbased on such markers, and commercializing/marketing diagnostics andservices utilizing such diagnostics

In one embodiment, the business methods herein comprise: establishingone or more markers using a method comprising: establishing a range ofconcentrations for said marker or markers in biological samples obtainedfrom a first population by measuring the concentrations of the marker ormarkers in the biological samples by detecting single molecules of themarker or markers; and commercializing the one or more markersidentified in the above step, e.g., in a diagnostic product. Thebiomarkers identified are preferably polypeptides or small molecules.Such polypeptides can be previously known or unknown. The diagnosticproduct herein can include one or more antibodies that specificallybinds to the marker (e.g., polypeptide).

In one embodiment, the business methods herein comprise: establishingone or more markers using a system comprising: establishing a range ofconcentrations for said marker in biological samples obtained from afirst population by measuring the concentrations of the marker thebiological samples by detecting single molecules of the marker; andproviding a diagnostic service to determine if an organism has or doesnot have a biological state or condition of interest. A diagnosticservice herein may be provided by a CLIA approved laboratory that islicensed under the business or the business itself. The diagnosticservices herein can be provided directly to a health care provider, ahealth care insurer, or a patient. Thus the business methods herein canmake revenue from selling e.g., diagnostic services or diagnosticproducts.

The business methods herein also contemplate providing diagnosticservices to, for example, health care providers, insurers, patients,etc. The business herein can provide diagnostic services by eithercontracting out with a service lab or setting up a service lab (underClinical Laboratory Improvement Amendment (CLIA) or other regulatoryapproval). Such service lab can then carry out the methods disclosedherein to identify if a particular marker or pattern of markers iswithin a sample.

The one or more markers are polypeptides or small molecules, or newchemical entities.

Kits

The invention further provides kits. In some embodiments, kits includean analyzer system and a label, as previously described. Kits of theinvention include one or more compositions useful for the sensitivedetection of a molecule, such as a marker, as described herein, insuitable packaging. In some embodiments, kits of the invention provide alabel, as described herein, together with other components such asinstructions, reagents, or other components. In some embodiments, thekit provides the label as separate components, in separate containers,such as an antibody and a fluorescent moiety, for attachment before useby the consumer. In some embodiments kits of the invention providebinding partner pairs, e.g., antibody pairs, that are specific for amolecule, e.g., a marker, where at least one of the binding partners isa label for the marker, as described herein. In some embodiments, thebinding partners, e.g., antibodies, are provided in separate containers.In some embodiments, the binding partners, e.g., antibodies, areprovided in the same container. In some embodiments, one of the bindingpartners, e.g., antibody, is immobilized on a solid support, e.g., amicrotiter plate or a paramagnetic bead. In some of these embodiments,the other binding partner, e.g., antibody, is labeled with a fluorescentmoiety as described herein.

Binding partners, e.g., antibodies, solid supports, and fluorescentlabels for components of the kits may be any suitable such components asdescribed herein.

The kits may additionally include reagents useful in the methods of theinvention, e.g., buffers and other reagents used in binding reactions,washes, buffers or other reagents for preconditioning the instrument onwhich assays will be run, filters for filtering reagents, and elutionbuffers or other reagents for running samples through the instrument.

Kits may include one or more standards, e.g., standards for use in theassays of the invention, such as standards of highly purified, e.g.,recombinant, protein markers, or various fragments, complexes, and thelike, thereof. Kits may further include instructions.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limiting the remaining disclosure.

Unless otherwise specified, processing samples in the Examples wereanalyzed in a single molecule detector (SMD) as described herein, withthe following parameters: Laser: continuous wave gallium arsenite diodelaser of wavelength 639 nm (Blue Sky Research, Milpitas, Calif.),focused to a spot size of approximately 2 microns (interrogation spaceof 0.004 pL as defined herein); flow rate=5 microliter/min through afused silica capillary of 100 micron square ID and 300 micron square OD;non-confocal arrangement of lenses (see, e.g., FIG. 1A); focusing lensof 0.8 numerical aperture (Olympus); silicon avalanche photodiodedetector (Perkin Elmer, Waltham, Mass.).

Example 1. Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)

The assay: The purpose of this assay was to detect the presence ofcardiac Troponin I (cTNI) in human serum. The assay format was atwo-step sandwich immunoassay based on a mouse monoclonal captureantibody and a goat polyconal detection antibody. Ten microliters ofsample were required. The working range of the assay is 0-900 pg/ml witha typical analytical limit of detection of 1-3 pg/ml. The assay requiredabout four hours of bench time to complete.

Materials: the following materials were used in the procedure describedbelow: Assay plate: Nunc Maxisorp, product 464718, 384 well, clear,passively coated with monoclonal antibody, BiosPacific A34440228P Lot #A0316 (5 μg/ml in 0.05 M sodium carbonate pH 9.6, overnight at roomtemperature); blocked with 5% sucrose, 1% BSA in PBS, and stored at 4°C. For the standard curve, Human cardiac Troponin I (BiosPacific Cat #J34000352) was used. The diluent for the standard concentrations washuman serum that was immonodepleted of endogenous cTNI, aliquoted andstored at −20° C. Dilution of the standards was done in a 96 well,conical, polypropylene, (Nunc product #249944). The following buffersand solutions were used: (a) assay buffer: BBS with 1% BSA and 0.1%TritonX-100; (b) passive blocking solution in assay buffer containing 2mg/ml mouse IgG, (Equitech Bio); 2 mg/ml goat IgG, (Equitech Bio); and 2mg/ml MAK33 poly, Roche#11939661; (c) detection Antibody (Ab): GoatPolyclonal antibody affinity purified to Peptide 3, (BiosPacific G129C),which was label with a fluorescent dye AlexaFluor 647, and stored at 4°C.; detection antibody diluent: 50% assay buffer, 50% passive blockingsolution; wash buffer: borate buffer saline Triton Buffer (BBST) (1.0 Mborate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); elutionbuffer: BBS with 4M urea, 0.02% Triton X-100 and 0.001% BSA.

Preparation of AlexaFluor 647 labeled antibodies: the detection antibodyG-129-C was conjugated to AlexaFluor 647 by first dissolving 100 ug ofG-129-C in 400 uL of the coupling buffer (0.1M NaHCO3). The antibodysolution was then concentrated to 50 μl by transferring the solutioninto YM-30 filter and subjecting the solution and filter tocentrifugation. The YM-30 filter and antibody was then washed threetimes by adding 400 ul of the coupling buffer. The antibody wasrecovered by adding 50□l to the filter, inverting the filter, andcentrifuging for 1 minute at 5,000×g. The resulting antibody solutionwas 1-2 ug/ul. AlexaFluor 647 NHS ester was reconstituted by adding 20ul DMSO to one vial of AlexaFluor 647, this solution was stored at −20°C. for up to one month. 3 ul of AlexaFluor 647 stock solution was addedto the antibody solution, which was then mixed and incubated in the darkfor one hour. After the one hour, 7.5 ul 1M tris was added to theantibody AlexaFluor 647 solution and mixed. The solution wasultrafiltered with YM-30 to remove low molecular weight components. Thevolume of the retentate, which contained the antibody conjugated toAlexaFluor 647, was adjusted to 200-400 □l by adding PBS. 3 ul 10% NaN3was added to the solution, the resulting solution was transferred to anUltrafree 0.22 centrifugal unit and spun for 2 minutes at 12,000×g. Thefiltrate containing the conjugated antibody was collected and used inthe assays.

Procedure: cTnI Standard and Sample Preparation and Analysis:

The standard curve was prepared as follows: working standards wereprepared (0-900 pg/ml) by serial dilutions of the stock of cTnI intostandard diluent or to achieve a range of cTnI concentrations of between1.2 pg/ml-4.3 μg/ml.

10 μl passive blocking solution and 10 μl of standard or of sample wereadded to each well. Standards were run in quadruplicate. The plate wassealed with Axyseal sealing film, centrifuged for 1 min at 3000 RPM, andincubated for 2 hours at 25° C. with shaking. The plate was washed fivetimes, and centrifuged until rotor reached 3000 RPM in an invertedposition over a paper towel. A 1 nM working dilution of detectionantibody was prepared, and 20 μl detection antibody were added to eachwell. The plate was sealed and centrifuged, and the assay incubated for1 hour at 25° C. with shaking. 30 μl elution buffer were added per well,the plate was sealed and the assay incubated for ½ hour at 25° C. Theplate was either stored for up to 48 hours at 4° C. prior to analysis,or the sample was analyzed immediately.

For analysis, 20 μl per well were acquired at 40 μl/minute, and 5 μlwere analyzed at 5 μl/minute. The data were analyzed based on athreshold of 4 sigma. Raw signal versus concentration of the standardswas plotted. A linear fit was performed for the low concentration range,and a non-linear fit was performed for the full standard curve. Thelimit of detection (LoD) was calculated as LOD=(3× standard deviation ofzeros)/slope of linear fit. The concentrations of the samples weredetermined from the equation (linear or non-linear) appropriate for thesample signal.

An aliquot was pumped into the analyzer. Individually-labeled antibodieswere measured during capillary flow by setting the interrogation volumesuch that the emission of only 1 fluorescent label was detected in adefined space following laser excitation. With each signal representinga digital event, this configuration enables extremely high analyticalsensitivities. Total fluorescent signal is determined as a sum of theindividual digital events. Each molecule counted is a positive datapoint with hundreds to thousands of DMC events/sample. The limit ofdetection the cTnI assay of the invention was determined by the mean+3SD method.

Results: Data for a typical cTnI standard curve measured inquadruplicate using the assay protocol is shown in Table 3.

TABLE 3 Standard Curve for cTnI cTnI Standard % (pg/ml) Signal DeviationCV 0 233 25 10.8 1.5625 346 31 8.9 3.125 463 35 7.5 6.25 695 39 5.6 12.51137 61 5.3 25 1988 139 7.0 50 3654 174 4.8 100 5493 350 6.4 200 8264267 3.2 400 9702 149 1.5 800 9976 50 0.5

The sensitivity of the analyzer system was tested in 15 runs and wasfound routinely to detect sub femtomol/l (fM) levels of calibrator, asshown by the data in Table 4. The precision was 10% at 4 and 12 pg/mlcTnI.

TABLE 4 Instrument Sensitivity Calibrator Signal (fM) counts CV 0 11 12302 9 60 1341 8 300 4784 7

Linearized standard curve for the range concentrations of cTnI are shownin FIG. 5.

The analytical limit of detection (LoD) was determined across 15sequential assays. The LoD was the mean of the 0 std+3 SD (n=4)intra-assay determinations. The average LoD was 1.7 pg/ml (range 0.4-2.8pg/ml).

The recovery of the sample was determined by analyzing samples of serumthat had been immunodepleted of cTnI and spiked with known amounts ofcTnI. Table 5 shows the data for sample recovery by the system analyzedover 3 days.

TABLE 5 Sample Recovery Spike Recovery Standard % (pg/ml) (mean)Deviation CV 5 5.7 0.9 16 15 13.7 0.2 2 45 43 0.6 2 135 151 6.2 4

The linearity of the assay was determined in pooled human serum that wasspiked with cTnI and diluted with standard diluent. The results in Table6 show the dilutions and % of the signal expected for the correspondingdilution.

TABLE 6 Assay Linearity Serum Dilution % of expected 1:2 79 1:4 87 1:896

These data show that the analyzer system of the invention allows forperforming highly sensitive laser-induced immunoassay for sub-femtomolarconcentrations of cTnI.

Example 2. Sandwich Bead-Based Assays for TnI

The assays described above use the same microtiter plate format wherethe plastic surface is used to immobilize target molecules. The singleparticle analyzer system also is compatible with assays done in solutionusing microparticles or beads to achieve separation of bound fromunbound entities.

Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained fromDynal (650.01-03, 10 mg/ml stock). Buffers use in the assay include: 10×borate buffer saline Triton Buffer (BBST) (1.0 M borate, 15.0 M sodiumchloride, 10% Triton X-100, pH 8.3); assay buffer (2 mg/ml normal goatIgG, 2 mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 MTris (pH 8.1), 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100,and 0.1% NaN3, stored at 4 C); and elution buffer (BBS with 4 M urea,0.02% Triton X-100, and 0.001% BSA, stored at 2-8 C). Antibodies used inthe sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific)with 1-2 biotins per IgG) and Det-Ab (G-129-C (BiosPacific) conjugatedto A647, 2-4 fluors per IgG). The standard is recombinant human cardiactroponin I (BiosPacific, cat # J34120352). The calibrator diluent is 30mg/ml BSA in TBS wEDTA.

Microparticles Coating: 100 ul of the MPs stock is placed in aneppendorf tube. The MPs are washed three times with 100 ul of BBST washbuffer by applying a magnet, removing the supernatant, removing themagnet, and resuspending in wash buffer. After the washes the MPs areresuspended in 100 ul of assay buffer and 15 ug of Bio-Ab are added. Themixture is then incubated for an hour at room temperature with constantmixing. The MPs are washed five times with 1 ml wash buffer as describedabove. After the washes the MPs are resuspended in 15 ml of assay buffer(or 100 ul to store at 4° C.).

Preparation of Standard and Samples: The standard is diluted withcalibrator diluent to prepare proper standard curve (usually 200 pg/mldown to 0.1 pg/ml). Frozen serum and plasma samples need to becentrifuged 10 minutes at room temperature at 13K rpm. Clarifiedserum/plasma is removed carefully to avoid taking any possible pelletsor floaters and put into fresh tubes. 50 ul of each standard or sampleis pippetted into appropriate wells.

Capture Target: 150 ul of MPs (after resuspension to 15 ml in assaybuffer+400 mM NaCl) are added to each well. The mixture is incubated onJitterBug, 5 at room temperature for 1 hr.

Washes and Detection: The plate is placed on a magnet and thesupernatant is removed after ensuring that all MPs are captured by themagnet. 250 ul of wash buffer are added after removing the plate fromthe magnet. The plate is then placed on the magnet and the supernatantis removed after ensuring that all MPs are captured by the magnet. 20 ulDet-Ab are added per well (Det-Ab to 500 ng/ml is diluted in assaybuffer+400 mM NaCl)). The mixture is incubated on JitterBug, 5 at roomtemperature for 30 min.

Washes and Elution: The plate is placed on a magnet and washed threetimes with wash buffer. The supernatant is removed after ensuring thatall MPs are captured by the magnet and 250 ul of wash buffer are added.After the washes the samples are transferred into a new 96-well plate.The new plate is then placed on the magnet and the supernatant isremoved after ensuring that all MPs are captured by the magnet. 250 ulof wash buffer are then added after removing the plate from the magnet.The plate is then placed on the magnet and the supernatant is removedafter ensuring that all MPs are captured by the magnet. 20 ul of elutionbuffer are then added and the mixture is incubated on JitterBug, 5 atroom temperature for 30 min.

Filter out MPs and transfer to 384-well plate: The standard and samplesare transferred into a 384-well filter plate placed on top of a 384-wellassay plate. The plate is then centrifuged at room temperature at 3000rpm with a plate rotor. The filter plate is removed and the appropriatecalibrators are added. The plate is covered and is ready to be run onSMD.

SMD: An aliquot is pumped into the analyzer. Individually-labeledantibodies are measured during capillary flow by setting theinterrogation volume such that the emission of only 1 fluorescentmolecule is detected in a defined space following laser excitation. Witheach signal representing a digital event, this configuration enablesextremely high analytical sensitivities. Total fluorescent signal isdetermined as a sum of the individual digital events. Each moleculecounted is a positive data point with hundreds to thousands of DMCevents/sample. The limit of detection the cTnI assay of the invention isdetermined by the mean+3 SD method.

Example 3. Concentration Range for cTnI in a Population of NormalNon-Diseased Subjects

A reference range or normal range for cTnI concentrations in human serumwas established using serum samples from 88 apparently healthy subjects(non-diseased). A sandwich immunoassay as described in Example 1 wasperformed and the number of signals or events as described above werecounted using the single particle analyzer system of the invention. Theconcentration of serum troponin I was determined by correlating thesignals detected by the analyzer with the standard curve as describedabove. All assays were perfumed in quadruplicate.

In accordance with recommendations by the current European and AmericanCardiology Societies (ESC/ACC) troponin assays should quantifyaccurately the 99th percentile of the normal range with an assayimprecision (CV) of less than 10% in order to distinguish reliablybetween patients with ACS and patients without ischemic heart disease,and risk stratification for adverse cardiac events. The assay showedthat the biological threshold (cutoff concentration) for TnI is at a TnIconcentration of 7 pg/ml, which is established at the 99th percentilewith a corresponding CV of 10% (FIG. 5). At the 10% CV level theprecision profile points at a TnI concentration of 4 and 12 pg/ml.

In addition, the assay correlates well with the Troponin-I standardmeasurements provided by the National Institute of Standards andTechnology (FIG. 6).

The assay of the invention is sufficiently sensitive and precise tofulfill the requirements of the ESC/ACC, and it is the most sensitiveassay for cardiac troponin I when compared to assays such as thosedescribed by Koerbin et al. (Ann Clin Biochem, 42:19-23 (2005). Theassay of the invention has a 10-20 fold greater sensitivity than thatcurrently available assays, which has determined the biologicalthreshold range to be 111-333 pg/ml cTnI.

Example 4. Detection of Early Release of TnI into the Circulation ofPatients with Acute Myocardial Infarction (AMI)

Study 1: 47 samples were obtained serially from 18 patients thatpresented with chest pain in the emergency department (ED). Thesepatients all had non-ST elevated ECG were, and were diagnosed with AMI.The concentration of cTnI in the initial samples from all 18 patientswas determined according to a commercial assay at the time of admissionto the emergency room to be <350 pg/ml (10% cutpoint), and 12 were <100pg/ml (99th %) percentile. These samples were tested at later timesusing the same commercial assay, and were determined to test positivefor cTnI. The same serum samples were also assayed for TnI according tothe assay of the invention as described in Examples 1 and 3, and theresults compared to the results obtained using the commercial assay.

Blood was drawn for the first time at the time the patient presentedwith chest pain (sample 1), and subsequently at intervals between 4-8hours (samples 2 at 12 hours; sample 3 at 16 hours; sample 4 at 24hours; sample 5 at 30 hours; sample 6 at 36 hours; sample 7 at 42 hours;and sample 8 at 48 hours). The serum was analyzed by the methods of theinvention and by a current commercial method, and the results obtainedare shown in FIG. 7. The analyzer of the invention detected TnI at thetime the patient presented with chest pain (sample 1), while thecommercial assay first detected cTnI at a much later time (sample 6 at36 hours). The concentration of TnI in sample 3 exceeded the biologicalthreshold level that was established using the analyzer of the invention(7 pg/ml, see FIG. 5), and indicated that sample 3 is positive for TnIto suggest the incidence of a cardiac event. The biological thresholdfor the commercial assay lies between 111 and 333 pg/ml of TnI.Accordingly, sample 3 would not have been considered to indicate apossible cardiac event.

In addition, the methods and compositions of the present invention allowfor much earlier diagnosis and possible intervention based on cardiactroponin levels, as evidenced by results for the first sample taken fromthe patients. In the 3 cases that had initial commercial assay cTnIvalues of between 100 and 350 ng/ml, all were positive for cTnI by theanalytical methods of the invention (i.e., cTnI over 7 pg/ml). In the 12cases that had initial commercial cTnI values of less than 100 pg/ml, 5were determined to be positive for a cardiovascular event according tothe assay of the invention (i.e., cTnI over 7 pg/ml). The prospectiveuse of the assay of the invention would have detected 53% more AMI casesthan the current commercial assay when the admission sample was tested.

Study 2: 50 additional serum samples, which tested negative according tothe commercial assay, were tested using the analyzer and assay of theinvention. The results are shown in FIG. 8. Of the 50 samples, 36 werewithin the 99th % and determined to be within the normal rangeestablished by the assay of the invention. However, the remaining 14samples that were determined to be within the commercial “normal” ornon-diseased range, tested above the biological threshold established bythe invention.

Therefore, the high sensitivity cTnI assay of the invention allows forthe detection of myocardial damage in patients when cTnI serum levelsare below threshold values by commercially available technology. The useof the highly sensitive and precise cTnI assay of the invention enablesdetection of AMI earlier than with existing cTnI assays, and therebyprovides the opportunity for appropriate diagnosis and early medicalintervention to improve the outcome.

Example 5. Detection of Leukotriene T4 (LTE4)

The assay was developed to quantify Leukotriene E₄ (LTE₄) in buffer as apreliminary assay for assays using, e.g., urine specimens. The assayformat was a one-step single antibody competitive immunoassay. Fiftymicroliters of sample were required. The typical working range of thisassay was 0-300 pg/ml with a typical limit of detection of 2-3 pg/ml(0.1-0.15 pg/sample). The assay required about four hours of bench timeto complete.

The following materials were prepared and used in the proceduredescribed below: Mouse anti-rabbit IgG coated plate provided in CaymanChemical Leukotriene E₄ (EIA Kit, Catalog #520411); stock LTE₄ Standard(purified LTE4 at 100 ng/ml in ethanol (Cayman Chemical Leukotriene E₄EIA Kit, Catalog #520411)); assay buffer (10×EIA buffer concentrate(Cayman Chemical Leukotriene E₄ EIA Kit, Catalog #520411)) diluted 1:10with 90 ml Nanopure water; buffer for dilution of standards (3%ethanol); anti-LTE₄ antibody (Leukotriene E₄ EIA antiserum (CaymanChemical Leukotriene E₄ EIA Kit, Catalog #520411) diluted with 30 ml EIAbuffer; streptavidin-Alexa detection reagent stock solution of 31(streptavidin labeled with AlexaFluor™ 647); tracer (LTE₄-biotinconjugate) was made compatible for detection by the analyzer; washbuffer (400× concentrate (Cayman Chemical Leukotriene E₄ EIA Kit,Catalog #520411)) diluted 1:40; elution buffer (borate buffered saline,pH 8.3 with 4M urea, 0.02% Triton X-100 and 0.001% BSA). The matrix ofthe tracer and the antiserum concentrations were tested to identify themost sensitive assay conditions.

A standard curve was prepared as follows: working standards wereprepared by making serial dilutions of the 100 ng/ml stock into assaybuffer to achieve a range of concentrations between 0.005 pg/ml and 3000pg/ml. 50 μl standard (or sample) were added per well of the assayplate. All standards were run in duplicate. Working tracer was preparedby diluting the tracer stock to 1 pg/ml with assay buffer. 50 μl tracer(or buffer) were added per well of the assay plate. A 10% workingantiserum solution was prepared by diluting 100% stock (made accordingto the kit instructions) into assay buffer. 50 μl antiserum (or buffer)were added per well of the assay plate; the plate was sealed andincubated overnight at 25° C. with shaking. A working streptavidin-Alexadetection reagent was prepared by diluting stock to 140 pM with assaybuffer. 15 ul of detection reagent were added to each well, and theplate was incubated for 30 min at 25° C. with shaking. The plate waswashed 5 times. 50 μl of elution buffer were added to each well, and theplate was incubated for ½ hour at 25° C. with shaking. The plate was useimmediately or stored for up to 48 hours at 4° C. prior to analysis.

20 μl were pumped into the analyzer at a rate of 40 μl/minute, and 5 μlof sample were analyzed at 5 μl/minute. The data files were analyzedusing a threshold=4 sigma, and a cross correlation of between 0-8 msec.Raw signal versus concentration was plotted for the standards, and alinear fit was used for low range standards, while a non-linear fit wasused for full standard curve. The limit of detection was calculated asLOD=80% of the maximum signal (no target control) (the concentration atwhich B/B₀=80%). The concentrations of samples were calculated from theequation (linear or non-linear) appropriate for the sample signal.

The competition curve of LTE4 is shown in FIG. 9. The LOD was calculatedto be 80% B/Bo=1.5 pg/ml (approximately 5 pM). The LTE4 assay performedusing a commercially available kit can detect LTE4 only if present at aconcentration of at least 30 pg/ml.

Therefore, the analyzer system can be used to detect levels of LTE4 toindicate the presence of an LTE4-related disorder e.g. asthma at theonset of disease, and alert clinicians to the need for therapeuticintervention at an early stage of the disease to improve the clinicaloutcome.

Example 6. Detection of Human Akt1

A sandwich immunoassay was developed for the quantification of lowlevels of Akt1 in serum samples. A standard curve was generated bydilution of a concentrated standard into a buffered protein solution.Ten microliters (μl) of assay buffer and 10 μl of sample or standardwere added to each well of a 384-well plate that had been coated with anantibody specific for Akt1 and incubated for two hours. Morespecifically antibody 841660 (R&D Systems) was coated onto Nunc Maxisorpplates @ 2.5 micrograms/ml. The plate was washed, and 20 μl of labeleddetection antibody specific for Akt1, AF1775 (R&D Systems), labeled withAlexaFluor 647, 2-4 fluors/IgG, was added to each well. After one hourof incubation the plate was washed to remove unbound detection antibody.Bound detection antibody was eluted and measured in the analyzerinstrument.

The following materials were used in the assay procedure describedbelow. Coated 384 well plate; assay buffer; resuspension buffer;dilution buffer; standard diluent; Akt1 standard; detection antibodyreagent for Akt1; wash buffer (10 mM Borate, 150 mM NaCl, 0.1%TritonX-100, pH 8.3); elution buffer (4 M urea with 0.02% Triton X-100and 0.001% BSA). Microplate shaker, set at “7”, Microplate washer, Platecentrifuge, Axyseal sealing film, Axygen product 321-31-051, Nuncpierceable sealing tape, Nunc product 235306.

Materials

Provided Reagents

Capture antibody: 841660 (R&D Systems), coated onto Nunc Maxisorp plates@ 2.5 micrograms/ml (384 well plate)

Assay buffer

Resuspension Buffer

Dilution Buffer

Standard diluent

Akt 1 standard

Detection antibody reagent for Akt1, AF1775 (R&D Systems), labeled withAlexaFluor 647, 2-4 fluors/IgG

Other Required Reagents

TritonX-100 Wash buffer (10 mM Borate, 150 mM NaCl, 0.1% TritonX-100, pH8.3)

Elution buffer (4 M urea with 0.02% Triton X-100 and 0.001% BSA)

Microplate shaker, set at “7”

Microplate washer

Plate centrifuge

Axyseal sealing film, Axygen product 321-31-051

Nunc pierceable sealing tape, Nunc product 235306

Procedure

Akt1 standard preparation

Resuspend standard in 0.5 ml Resuspension Buffer, finalconcentration=170 ng/ml

Dilute standard 1:3 in Dilution Buffer=57 ng/ml

Dilute standard 1: 19 in Standard Diluent=3 ng/ml

Do serial 3 fold dilutions down to 4.1 pg/ml in Standard Diluent

Add 10 μl Assay Buffer per well

Add 10 μl standard or sample per well

Seal plate with Axyseal sealing film

Spin 1 min at 3000 RPM

Incubate 2 hours at 25° C. with shaking

Wash plate five times

Spin plate inverted on a paper towel 1 min at 3000 RPM

Add 20 μl detection antibody reagent per well

Seal plate with Axyseal sealing film

Spin plate inverted on a paper towel 1 min at 3000 RPM

Incubate 1 hour at 25° C. with shaking

Wash plate five times

Spin plate inverted on a paper towel 1 min at 3000 RPM

Add 30 μl elution buffer per well

Spin 1 min at 3000 RPM

Seal with Nunc pierceable sealing tape, secure tight seal with roller

Incubate ½ hour at 25° C. with shaking

The plate may be stored for up to 48 hours at 4° C. prior to analysis

Analyze on Zeptx instrument

The Akt1 standard curve was generated as follows. Akt1 standards wereprepared to achieve a range of between 4.1 pg/ml to 170 ng/ml Akt1. 10μl of each standard dilution (or sample) were added to the assay platewells. The plate was sealed and incubated for 2 hours at 25° C. withshaking. The plate was washed and centrifuged dry. 20 μl detectionantibody reagent was added per well and incubated for 1 hour at 25° C.with shaking. The antibody-Akt1 complex was disrupted by adding 30 μlelution buffer per well and incubating for ½ hour at 25° C. withshaking. The plate was either used immediately or stored for up to 48hours at 4° C. prior to analysis. Eluate was pumped into the analyzer.

Data for a typical Akt1 standard curve measured in quadruplicate usingthe assay protocol is given in Table 14, and the graphed data is shownin FIG. 10.

TABLE 14 Standard curve for Akt1 Concentration Akt1 standard AverageStandard % (pg/ml) Signal deviation CV 0 113 16 14 4.1 126 10 8 12.4 1331 0 37 151 34 22 111 173 15 8 333 350 74 21 1000 733 136 19 3000 1822243 13

Intra-Assay Precision was tested using 36 replicate samples of the 1000pg/ml standard by assaying the samples on a single plate. The averagesignal was 1822±243 with a % CV=13. The limit of detection of the assay(LoD) was determined by adding two standard deviations to the meansignal of thirty six zero standard replicates and calculating thecorresponding Akt1 concentration from the standard curve. The LoD wascalculated to be 25 pg/ml.

Therefore, the analyzer system can be used to detect levels of Akt1 todetermine the presence or absence of an Akt1-related disorder, e.g.cancer.

Example 7. Detection of TGF-β

A sandwich immunoassay was developed for the quantification of lowlevels of TGFβ in serum. A standard curve was generated by dilution of aconcentrated standard into a buffeted protein solution. Ten microliters(μl) of assay buffer and 10 μl of sample or standard were added to eachwell of a 384-well plate coated with an antibody specific for TGFβ andincubated for two hours. The plate was washed and 20 μl of labeleddetection antibody specific for TGFβ was added to each well. After onehour of incubation the plate was washed to remove unbound detectionantibody. Bound detection antibody was eluted and measured in theanalyzer instrument.

The following materials were used in the assay procedure describedbelow. Coated 384 well plate; assay buffer; standard diluent; 10 ug/mlstock solution of TGFβ standard; detection antibody reagent for TGFβ;TritonX-100 Wash buffer (10 mM Borate, 150 mM NaCl, 0.1% TritonX-100, pH8.3); elution buffer (4 M urea with 0.02% Triton X-100 and 0.001% BSA).

The TGF-β standard curve was generated as follows. TGF-β standards wereprepared to achieve a range of between 100 ng/ml to 4.1 pg/ml TGFβ. 10μl assay buffer and 10 μl standard or sample were added to each well.The plate was sealed and incubated for 2 hours at 25° C. with shaking.The plate was sealed and incubated for 2 hours at 25° C. with shaking.The plate was washed and centrifuged dry. 20 μl detection antibodyreagent was added per well and incubated for 1 hour at 25° C. withshaking. The antibody-TGF-β complex was disrupted by adding 30 μlelution buffer per well and incubating for ½ hour at 25° C. withshaking. The plate was either used immediately or stored for up to 48hours at 4° C. prior to analysis. Eluate was pumped into the analyzer.

Data for a typical TGF-β standard curve measured in quadruplicate usingthe assay protocol is given in Table 15, and the graphed data is shownin FIG. 11.

TABLE 15 Standard curve for TGF-β Concentration Average Standard %(pg/ml) Signal deviation CV 0 1230 114 9 4 1190 68 6 12 1261 132 10 371170 158 14 111 1242 103 8 333 1364 135 10 1000 1939 100 5 3000 3604 49714

The limit of detection of the assay (LoD) was determined by adding twostandard deviations to the mean signal of twenty zero standardreplicates and calculating the corresponding TGFβ concentration from thestandard curve. The LoD=350 pg/ml.

Therefore, the analyzer system can be used to detect levels of TGFβ todetermine the presence or absence of an TGFβ-related disorder, e.g.cancer.

Example 8. Detection of Fas Ligand

A sandwich immunoassay for the quantification of low levels of Fasligand in serum. A standard curve was generated by dilution of aconcentrated standard into a buffered protein solution. Ten microliters(μl) of assay buffer and 10 μl of sample or standard were added to eachwell of a 384-well plate coated with an antibody specific for Fas ligandand incubated for 2 hours. The plate was washed and 20 μl of labeleddetection antibody specific for Fas ligand was added to each well. Aftera 1 hour incubation the plate was washed to remove unbound detectionantibody. Bound detection antibody was eluted and measured in theZept_(x) instrument.

The Fas ligand standard curve was generated as follows. Fas ligandstandards were prepared to achieve a range of between 100 ng/ml to 4.1pg/ml Fas ligand. 10 μl assay buffer and 10 μl standard or sample wereadded to each well. The plate was sealed and incubated for 2 hours at25° C. with shaking. The plate was sealed and incubated for 2 hours at25° C. with shaking. The plate was washed and centrifuged dry. 20 μldetection antibody reagent was added per well and incubated for 1 hourat 25° C. with shaking. The antibody-Fas ligand complex was disrupted byadding 30 μl elution buffer per well and incubating for ½ hour at 25° C.with shaking. The plate was either used immediately or stored for up to48 hours at 4° C. prior to analysis.

Data for a typical Fas ligand standard curve measured in quadruplicateusing the assay protocol is given in Table 16.

TABLE 16 Standard curve for Fas ligand Concentration Fas AverageStandard % ligand standard (pg/ml) Signal deviation CV 0 935 82 9 1.21007 44 4 3.4 1222 56 5 11 1587 70 4 33 2869 52 2 100 5939 141 2 3009276 165 2 900 11086 75 1Infra-Assay Precision was tested using 12 replicate samples of 3standard concentrations by assaying the samples on a single plate. Themean, standard deviation and CV for the 12 values for each of the threepoints are shown in Table 17.

TABLE 17 Intra-assay precision for Fas ligand Concentration AverageStandard % (pg/ml) Signal deviation CV 11 1717 128 7 33 3031 262 9 1006025 257 4

The limit of detection of the assay (LoD) was determined by adding twostandard deviations to the mean signal of twenty zero standardreplicates and calculating the corresponding Fas ligand concentrationfrom the standard curve. The LoD was calculated to be 2.4 pg/ml

Therefore, the analyzer system of the invention can detect levels of Fasligand to indicate the presence of a Fas ligand-related disorder e.g.cancer, allograft rejection and degenerative diseases such asosteoarthritis.

Example 9. Sandwich Assays for Biomarker TREM-1

Recent reports have established TREM-1 as a biomarker of bacterial orfungal infections (see, e.g., Bouchon et al. (2000) J. Immunol.164:4991-5; Colonna (2003) Nat. Rev. Immunol. 3:445-53; Gibot et al.(2004) N. Engl. J. Med. 350:451-8; Gibot et al. (2004) Ann. Intern. Med.141:9-15. Assays for TREM-1 have been developed using a sandwich assayformat (Sandwich Assay for Detection of Individual Molecules, U.S.Provisional Patent Application No. 60/624,785). Assay reagents forTREM-1 detection are available commercially (R&D Systems, Minneapolis,Minn.). The assay was done in a 96 well plate. A monoclonal antibody wasused as the capture reagent, and either another monoclonal or apolyclonal antibody was used for detection. The detection antibody waslabeled with AlexaFluorA647®.

The assay protocol was as follows:

1. Coat plates with the capture antibody, washed 5×,

2. Block in 1% BSA, 5% sucrose in PBS,

3. Add the target diluted in serum, incubate, wash 5×,

4. Add the detection antibody, incubate, wash 5×

5. Add 0.1 M glycine pH 2.8 to release the bound assay components fromthe plate.

6. Transfer samples from the processing plate to the detection plate,bring the pH of the sample to neutral and run on the single particleanalyzer system.

FIG. 13 shows a standard curve of TREM-1 generated using the assay. Theassay was linear in the measured range of 100-1500 femtomolar. An ELISAassay from R&D Systems has recently been introduced. The standard curvereported for their ELISA assay is between 60-4000 pg/ml. This Examplesuggests we can routinely measure 100 fM (4.7 pg/ml) in a standardcurve, allowing for about 10× more sensitive measurements. In addition,standard curves for chemokines, T cell activation molecules, celladhesion molecules and signal transduction molecules have been generated(FIG. 15). The results show that the detection by the detection ofanalyte using the single particle analyzer is consistently between 10and 100 fold more sensitive than detection using ELISA assays.

Example 10. Sandwich Assays for Biomarkers: IL-6 and IL-8 Levels inSerum

The Assay:

This protocol describes a sandwich immunoassay for the quantification oflow levels of IL-6 in serum using the single particle analyzer system ofthe invention. A standard curve was generated by dilution of aconcentrated standard into a buffered protein solution. Ten microliters(μl) of assay buffer and 10 μl of sample or standard were added to eachwell of a 384-well plate coated with an antibody specific for IL-6 andincubated for two hours. The plate was washed, and 20 μl of labeleddetection antibody specific for IL-6 was added to each well. After onehour of incubation the plate was washed to remove unbound detectionantibody. Bound detection antibody was eluted and measured in the singleparticle analyzer instrument.

Materials: the following materials were used in the procedure describedbelow: coated 384 well plate; assay buffer; standard diluent; 100 ng/mlstock solution of IL-6 standard; detection antibody for IL-6 (R&DSystems) labeled with AlexaFluor 647 dye; TritonX-100 Wash buffer (10 mMBorate, 150 mM NaCl, 0.1% TritonX-100, pH 8.3); Elution buffer (4 M ureawith 0.02% Triton X-100 and 0.001% BSA); Microplate shaker, set at “7”;Microplate washer; Plate centrifuge; Axyseal sealing film, Axygenproduct 321-31-051; and Nunc pierceable sealing tape, Nunc product235306.

Procedure:

IL-6 Standard and Sample Preparation and Analysis

A standard curve for IL-6 was prepared as follows: 100 ng/ml stocksolution were thawed, and the stock solution was diluted 1:1000 to 100pg/ml in standard diluent by doing six serial, 3 fold dilutions toobtain a range of concentration having the lowest standard concentrationof 0.14 pg/ml. 10 μl assay buffer and 10 μl standard or sample wereadded to each well per well of the coated 384 well plate. The plate wassealed with Axyseal sealing film, and centrifuged for one minute at 3000RPM. The assay plate was incubated for 2 hours at 25° C. with shaking;washed five times; and centrifuged while inverted on a paper towel forone minute at 3000 RPM. 20 μl detection antibody reagent was added toeach well; the plate was sealed with Axyseal sealing film, andcentrifuged for one minute at 3000 RPM. The assay plate was incubatedfor one hour at 25° C. with shaking, washed five times, and centrifugedwhile inverted on a paper towel for one minute at 3000 RPM. 30 μlelution buffer was added to each well; the plate was sealed with Nuncpierceable sealing tape, and a tight seal was secured using with roller.The assay plate was centrifuged for one minute at 3000 RPM, andincubated for ½ hour at 25° C. with shaking. Analysis of the assay wasperformed immediately. Alternatively, the plate was stored for up to 48hours at 4° C. prior to analysis.

Samples of serum from EDTA treated whole blood of 32 blood bank donorswere analyzed for IL-6.

Results:

Data for a typical IL-6 standard curve measured in quadruplicate usingthe assay protocol is shown in Table 18.

TABLE 18 Standard Curve for IL-6 Concentration Average Standard (pg/ml)Signal deviation CV 370 11035 206 2% 125 9983 207 2% 41 8522 95 1% 145023 108 2% 4.5 2577 124 5% 1.7 1178 114 10%  0.5 577 36 6% 0 106 1514% 

Linearized standard curves for higher and low range concentrations ofIL-6 are shown in FIGS. 15 A-B, respectively. The assay allowed fordetection of IL-6 at less than 0.5 pg/ml (FIGS. 14 A-B). The limit ofdetection (LoD) was calculated to be 0.06 pg/ml. The limit of detectionof the assay (LoD) was determined by adding two standard deviations tothe mean signal of the zero standard replicates and calculating thecorresponding IL-6 concentration from the standard curve. This level ofsensitivity is excellent for detection of even normal levels of IL-6which range between 0.5 and 10 pg/ml.

Assays to detect IL-6 and IL-8 in serum of blood samples from blood bankdonors were performed, and the results of the analysis are shown inFIGS. 14 C and D. IL-6 was quantified in 100% of the samples (32/32).The average concentration of IL-6 was 2.3 pg/ml, and the range ofconcentration was 0.2 to >26 pg/ml (FIG. 14 C). The same samples werealso assayed for IL-8 essentially using the procedure described forIL-6. IL-8 standards and IL-8 specific antibodies were used. A standardcurve for IL-8 was established (not shown) and used to determine theconcentration of IL-8 in the samples (FIG. 14D). IL-8 was quantified in100% (32/32) samples. The average concentration for IL-8 was 7.3 pg/ml,and the range of concentration was 1.2 to >26 pg/ml.

Measurements of IL-6 or any particle of interest can be measured at lowand higher concentrations (FIGS. 14 A and B) by switching the detectionof the analyzer from counting molecules (digital signal) to detectingthe sum of photons (analog signal) that are generated at the higherconcentrations of analyte. This is shown in a general way in FIG. 14E.The single particle analyzer has an expanded linear dynamic range of 6logs. The ability to increase the dynamic range for detecting theconcentration of a particle in a sample allows for the determination ofthe concentration of a particle for normal (lower concentration range)and disease levels (higher concentration range). The range of detectionfor normal and disease levels of IL-6 is shown in FIG. 15F.

What is claimed is:
 1. An analyzer comprising: a) an electromagneticradiation source for stimulating a fluorescent moiety; b) a capillaryflow cell for passing said label; c) a source of motive force for movingsaid label in said capillary flow cell; d) an interrogation spacedefined within said capillary flow cell for receiving electromagneticradiation emitted from said electromagnetic source; and e) anelectromagnetic radiation detector operably connected to saidinterrogation space for measuring an electromagnetic characteristic of astimulated fluorescent moiety; a processor operatively connected to thedetector, wherein the processor is configured to execute instructionsstored on a non-transitory computer-readable medium, and wherein theinstructions, when executed by the processor, cause the processor to:determine a threshold photon value corresponding to a background signalin the interrogation space, determine the presence of a fluorescentmoiety in the interrogation space in each of a plurality of bins byidentifying bins having a photon value greater than the threshold value,and compare the number of bins having a photon value greater than thethreshold value to a standard curve.
 2. The analyzer system of claim 1,wherein said electromagnetic radiation source is a laser, and whereinsaid laser has a power output of at least about 3, 5, 10, or 20 mW. 3.The analyzer system kit of claim 1, wherein the analyzer comprises notmore than one interrogation space.
 4. The analyzer system of claim 1wherein said electromagnetic radiation source is a continuous waveelectromagnetic radiation source.
 5. The analyzer system of claim 4,wherein said continuous wave electromagnetic radiation source is alight-emitting diode or a continuous wave laser.
 6. The analyzer systemof claim 1, wherein said motive force is pressure.
 7. The analyzersystem of claim 1, wherein said detector is an avalanche photodiodedetector.
 8. The system of claim 1, further comprising a confocaloptical arrangement for deflecting a laser beam onto said interrogationspace and for imaging a stimulated fluorescent moeity, wherein saidconfocal optical arrangement comprises an objective lens having anumerical aperture of at least about 0.8.
 9. The analyzer system ofclaim 1 further comprising a sampling system capable of automaticallysampling a plurality of samples and providing a fluid communicationbetween a sample container and said interrogation space.
 10. Theanalyzer system of claim 1 further comprising a sample recovery systemin fluid communication with said interrogation space, wherein saidrecovery system is capable of recovering substantially all of saidsample.